Antibodies that bind to EphA2 and methods of use thereof

ABSTRACT

Provided herein is disclosure about the development and characterization of an antibody that binds to antigen EphA2 which is present on a variety of human cancers from breast, lung, prostate, and colons. Methods of diagnosing and treating various cancers by using such antibodies directed against this antigen are also disclosed.

TECHNICAL FIELD

This invention is in the fields of biology and immunotherapy. Morespecifically, it concerns the discovery of a family of antibodies thatbind to EphA2. The invention further provides the diagnosis and/ortreatment of a variety of human diseases and cancers associated withEphA2 using anti-EphA2 family antibodies.

BACKGROUND OF THE INVENTION

In addition to their known uses in diagnostics, antibodies have beenshown to be useful as therapeutic agents. For example, immunotherapy, orthe use of antibodies for therapeutic purposes has been used in recentyears to treat cancer. Passive immunotherapy involves the use ofmonoclonal antibodies in cancer treatments. See for example, Cancer:Principles and Practice of Oncology, 6^(th) Edition (2001) Chapt. 20 pp.495-508. These antibodies can have inherent therapeutic biologicalactivity both by direct inhibition of tumor cell growth or survival andby their ability to recruit the natural cell killing activity of thebody's immune system. These agents can be administered alone or inconjunction with radiation or chemotherapeutic agents. Rituximab andTrastuzumab, approved for treatment of non-Hodgkin's lymphoma and breastcancer, respectively, are two examples of such therapeutics.Alternatively, antibodies can be used to make antibody conjugates wherethe antibody is linked to a toxic agent and directs that agent to thetumor by specifically binding to the tumor. Gemtuzumab ozogamicin is anexample of an approved antibody conjugate used for the treatment ofleukemia. Monoclonal antibodies that bind to cancer cells and havepotential uses for diagnosis and therapy have been disclosed inpublications. See, for example, the following patent applications whichdisclose, inter alfa, some molecular weights of target proteins: U.S.Pat. No. 6,054,561 (200 KD c-erbB-2 (Her2), and other unknown antigens40-200 KD in size) and U.S. Pat. No. 5,656,444 (50 KD and 55 KD,oncofetal protein). Example of antibodies in clinical trials and/orapproved for treatment of solid tumors include: Trastuzumab (antigen:180 kD, HER2/neu), Edrecolomab (antigen: 40-50 kD, Ep-CAM), Anti-humanmilk fat globules (HMFG1) (antigen >200 kD, HMW Mucin), Cetuximab(antigens: 150 kD and 170 kD, EGF receptor), Alemtuzumab (antigen: 21-28kD, CD52), and Rituximab (antigen: 35 kD, CD20).

The antigen targets of trastuzumab (Her-2 receptor), which is used totreat breast cancer, and cetuximab (EGF receptor), which is in clinicaltrials for the treatment of several cancers, are present at somedetectable level on a large number of normal human adult tissuesincluding skin, colon, lung, ovary, liver, and pancreas. The margin ofsafety in using these therapeutics is possibly provided by thedifference in the level of expression or in access of or activity of theantibody at these sites.

Another type of immunotherapy is active immunotherapy, or vaccination,with an antigen present on a specific cancer(s) or a DNA construct thatdirects the expression of the antigen, which then evokes the immuneresponse in the individual, i.e., to induce the individual to activelyproduce antibodies against their own cancer. Active immunization has notbeen used as often as passive immunotherapy or immunotoxins.

Several models of disease (including cancer) progression have beensuggested. Theories range from causation by a singleinfective/transforming event to the evolution of an increasingly“disease-like” or ‘cancer-like’ tissue type leading ultimately to onewith fully pathogenic or malignant capability. Some argue that withcancer, for example, a single mutational event is sufficient to causemalignancy, while others argue that subsequent alterations are alsonecessary. Some others have suggested that increasing mutational loadand tumor grade are necessary for both initiation as well as progressionof neoplasia via a continuum of mutation-selection events at thecellular level. Some cancer targets are found only in tumor tissues,while others are present in normal tissues and are up-regulated and/orover-expressed in tumor tissues. In such situations, some researchershave suggested that the over-expression is linked to the acquisition ofmalignancy, while others suggest that the over-expression is merely amarker of a trend along a path to an increasing disease state.

One aspect required for the ideal diagnostic and/or therapeutic antibodyis the discovery and characterization of an antigen that is associatedwith a variety of cancers. There are few antigens that are expressed ona number of types of cancer (e.g., “pan-cancer” antigen) that havelimited expression on non-cancerous cells. The isolation andpurification of such an antigen would be useful for making antibodies(e.g., diagnostic or therapeutic) targeting the antigen. An antibodybinding to the “pan-cancer” antigen could be able to target a variety ofcancers found in different tissues in contrast to an antibody against anantigen associated with only one specific type of cancer. The antigenwould also be useful for drug discovery (e.g., small molecules) and forfurther characterization of cellular regulation, growth, anddifferentiation.

What is needed are novel targets on the surface of diseased and/orcancer cells that may be used to treat such diseases and/or cancers withantibodies and other agents which specifically recognize the cellsurface targets. There exists a further need, based on the discoveriesdisclosed herein, for novel antibodies and other agents whichspecifically recognize targets on the surface of cells that canmodulate, either by reducing or enhancing, the disease-promotingactivities of EphA2.

EphA2, previously known as ECK, is a 130 kD transmembrane receptortyrosine kinase that is expressed on adult epithelial cells. SeeLindberg, et al. (1990) Mol. Cell. Biol. 10: 6316-6324. EphA2 is onemember of the Eph family of receptor tyrosine kinases, which are uniquein that they recognize ligands, known as ephrins, which are anchored tothe membrane of adjacent cells. See Bartley, et al. (1994) Nature 368:558-560. The sequence of the human receptor EphA2 is known in theliterature. It encompasses an extracellular domain of 534 amino acids, atransmembrane domain of 24 amino acids, and a cytoplasmic domain of 418amino acids that contains the tyrosine kinase domain.

EphA2 is over-expressed in a large number of cancer cells, for example,in breast, prostate, lung, and colon carcinomas, and in aggressivemelanomas, but reportedly is not over-expressed in non-cancerous lesionsof these same tissues. See, for example, Rosenberg, et al. (1997) Am. J.Physiol. 273: G824-G832; Easty, et al., (1995) Int. J. Cancer 60:129-136; Walker-Daniels, et al. (1999) Prostate 41: 275-280; Zantek etal. (1999) Cell Growth & Differ. 10: 629-638; Zantek et al. (2001) Clin.Cancer Res. 7: 3640-3648; Zelniski et al. (2001) Cancer Res. 61:2301-2306; WO 01/121172; and WO 01/12840. Moreover, cells that have beentransformed to over-express EphA2 demonstrate malignant growth andligand binding, which causes EphA2 to be internalized and degraded, andreverses the oncogenic effect of EphA2 over-expression. See Zelniski etal. (2001) Cancer Res. 61: 2301-2306; and Walker-Daniels, et al. (2002)Mol. Cancer Res. 1: 79-87.

Using EphA2 as a therapeutic target has been proposed by others in theart. One author suggested using EphA2 ligands such as an Ephrin A1fusion to human immunoglobulin G. Exposure of cells expressing EphA2 tothe Ephrin A1-Fc fusion protein resulted in a down-regulation of EphA2expression. See Duxbury, et al. (2004) BBRC 320:1096-1102.

Antibodies to eck/EphA2 are known, see e.g., Lindberg, et al. (1990)Mol. Cell. Biol. 10: 6316-6324. The use of antibody-based targetingusing anti-EphA2 antibodies has been described by others, see e.g.,Charles-Kinch et al. (2002) Cancer Res. 62:2840-2847, which describesusing extracellular domain of EphA2 fused to human immunoglobulin togenerated monoclonal antibodies against EphA2. Treatment of cancer cellswith these anti-EphA2 monoclonal antibodies resulted in morphologicalchanges and inhibition of cell growth on soft agar.

Antibodies to EphA2 have been made and proposed to be useful in thetreatment of cancer (see e. g., U.S. Pat. No. 6,927,203; InternationalPatent Publication Nos. WO01/12840 and WO01/12172; U.S. ProvisionalPatent Application Nos. 60/379,322 and 60/379,368; U.S. Pat. No.5,824,303). WO2003US15044 describes methods comprising theadministration of an effective amount of an antibody that binds to EphA2and agonizes EphA2, thereby increasing EphA2 phosphorylation anddecreasing EphA2 levels. In other embodiments, that applicationdescribes the administration of an effective amount of an antibody thatbinds to EphA2 and inhibits cancer cell colony formation in soft agar,inhibits tubular network formation in three-dimensional basementmembrane or extracellular matrix preparation, preferentially binds to anEphA2 epitope that is exposed on cancer cells but not non-cancer cells,and/or has a low Koff, thereby, inhibiting tumor cell growth andmetastasis. U.S. Pat. No. 6,927,203 describes antibodies that impedeproliferation of tumor cells using an antibody that increases thephosphotyrosine content of EphA2.

PCT patent application WO 200391383 describes peptides derived fromEphA2 and their use in anti-tumor immunotherapy. It describes peptidevaccination or immunotherapy based on an EphA2 epitope that may be usedto induce or mimic a cytotoxic T lymphocyte cell response to tumor cellsthat over-express EphA2.

While EphA2 antibodies are known, there remains a need for particularanti-EphA2 antibodies that are extremely effective in inhibiting tumorcell growth, beyond the level of effectiveness shown by the EphA2antibodies shown in the prior art.

All references, publications, and patent applications disclosed hereinare hereby incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

The invention disclosed herein concerns antibodies that bind to anantigen, EphA2, which is expressed on a variety of human cancers.Accordingly, in one aspect, the invention is a family of antibodies, oran antibody or a polypeptide (which may or may not be an antibody) thatbinds preferentially to the antigen EphA2. The antibodies claimed in thepresent invention are surprisingly and very effective in inhibitingtumor cell growth, beyond the level of effectiveness shown by the EphA2antibodies shown in the prior art. Some of these antibodies are referredto herein as LUCA19, SPL1, LUCA40, or SG5. In aspects of this invention,certain antibodies have been discovered that have the ability to impedeproliferation of tumor cells while not increasing the phosphotyrosinecontent of EphA2.

In another aspect, the invention is a monoclonal antibody anti-EphA2that is produced by any one of the following host cell lines: PTA-5070deposited Mar. 20, 2003 at the American Type Culture Collection,PTA-6056 deposited Jun. 8, 2004 at the American Type Culture Collection,PTA-6059 deposited Jun. 8, 2004 at the American Type Culture Collection,and an SG5-producing host cell line (SG.3.15.B4.1F9) was deposited atthe American Type Culture Collection on Feb. 2, 2006.

In another aspect, the invention is an antibody or a polypeptide (whichmay or may not be an antibody) that competitively inhibits specificbinding of the anti-EphA2 antibody to EphA2. In another aspect, theinvention is an antibody or a polypeptide (which may or may not be anantibody) that binds preferentially to the same epitope on EphA2 asLUCA19, SPL1, SG5 or LUCA40 binds preferentially.

In another aspect, the invention is an antibody comprising a fragment ora region of the anti-EphA2 antibody. In one embodiment, the fragment isa light chain of the anti-EphA2 antibody. In another embodiment, thefragment is a heavy chain of the anti-EphA2 antibody. In yet anotherembodiment, the fragment contains one or more variable regions from alight chain and/or a heavy chain of the anti-EphA2 antibody. In yetanother embodiment, the fragment contains one or more complementaritydetermining regions (CDRs) from a light chain and/or a heavy chain ofthe anti-EphA2 antibody.

In another aspect, the invention provides polypeptides (which may or maynot be antibodies) comprising any of the following: a) one or more CDRs;b) three CDRs from the light chain; c) three CDRs from the heavy chain;d) three CDRs from the light chain and three CDRs from the heavy chain;e) the light chain variable region; f) the heavy chain variable regionof the anti-EphA2 antibody.

In another aspect, the invention is a humanized antibody derived fromLUCA19, SPL1, SG5 or LUCA40. In some embodiments, the humanized antibodycomprises one or more CDRs of the anti-EphA2 antibody. In anotheraspect, the invention provides a humanized antibody that binds to thesame epitope(s) as anti-EphA2 antibody. Generally, a humanized antibodyof the invention comprises one or more (one, two, three, four, five,six) CDRs that are the same and/or derived from the CDR(s) of anti-EphA2antibody. In other aspect, the invention provides a human antibody thatbinds to the same epitope(s) on EphA2 as anti-EphA2 antibody.

In anther aspect, the invention is a chimeric antibody comprisingvariable regions derived from variable regions of a heavy chain and alight chain of anti-EphA2 antibody and constant regions derived fromconstant regions of a heavy chain and a light chain of a human antibody.

In yet another aspect, the invention is a host cell (ATCC No. PTA-5070,PTA-6056, PTA-6059, or SG.3.15.B4.1F9 (ATCC No. PTA-XXXX)) or progenythereof which produces monoclonal anti-EphA2 antibody.

In another aspect, the invention is an isolated polynucleotide thatencodes for anti-EphA2 antibody that is produced by a host cell with adeposit number of ATCC No. PTA-5070, PTA-6056, or PTA-6059 or host cellSG.3.15.B4.1F9 (ATCC No. PTA-XXXX) or progeny thereof. In anotheraspect, the invention provides polynucleotides encoding any of theantibodies (including antibody fragments) as well as any otherpolypeptides described herein.

In another aspect, the invention is a complex of EphA2 bound by any ofthe antibody or polypeptides described herein. In some embodiments, theEphA2 is present on melanomas, breast, colon, lung, or prostate cancercells. In some embodiments, the invention is a complex of EphA2 bound byanti-EphA2 antibody or an antibody that binds preferentially to theepitope that anti-EphA2 antibody binds preferentially. In oneembodiment, anti-EphA2 antibody or any antibody that bindspreferentially to the epitope that anti-EphA2 antibody bindspreferentially is linked to a therapeutic agent (such as a toxin).

In another aspect, the invention is a complex of a cancer cellexpressing EphA2 bound by any of the antibody or polypeptides describedherein. In some embodiments, the cancer cell is melanomas, breast,colon, lung, or prostate cancer cell. In some embodiments, the antibodyis SPL1, LUCA19, SG5 or LUCA40 or any antibody that binds preferentiallyto an epitope that any of these anti-EphA2 antibodies bindpreferentially. In some embodiments, SPL1, LUCA19, SG5 or LUCA40antibody or any antibody that binds preferentially to an epitope thatany of these anti-EphA2 antibodies bind preferentially is linked to atherapeutic agent (such as a toxin).

In another aspect, the invention is a complex of an epitope on EphA2that SPL1, LUCA19, SG5 or LUCA40 preferentially binds, which is in turnbound by any of the antibody or polypeptides described herein. In someembodiments, the epitope is on melanomas, breast, colon, lung, orprostate cancer cell. In some embodiments, the antibody is SPL1, LUCA19,SG5 or LUCA40 or any antibody that binds preferentially to an epitopethat any of these anti-EphA2 antibodies bind preferentially. In someembodiments, SPL1, LUCA19, SG5 or LUCA40 or any antibody that bindspreferentially to an epitope that any of these anti-EphA2 antibodiesbind preferentially is linked to a therapeutic agent (such as a toxin).

In another aspect, the invention is a pharmaceutical compositioncomprising any of the polypeptides (including any of the antibodies suchas anti-EphA2 antibody) or polynucleotides described herein, such aspharmaceutical compositions comprising the anti-EphA2 antibody, theanti-EphA2 antibody linked to a therapeutic agent, an antibodycomprising a fragment of the anti-EphA2 antibody, a humanized antibodyof the anti-EphA2 antibody, a chimeric antibody comprising variableregions derived from variable regions of the anti-EphA2 antibody andconstant regions derived from constant regions of a human antibody, or ahuman antibody with one or more properties of the anti-EphA2 antibody,and a pharmaceutically acceptable excipient.

In yet another aspect, the invention is a method of generatingmonoclonal antibody anti-EphA2 reactive with diseased and/or cancerouscells comprising the steps of: (a) immunizing a host mammal with animmunogen; (b) obtaining lymphocytes from the mammal; (c) fusinglymphocytes (b) with a myeloma cell line to produce a hybridoma; (d)culturing the hybridoma of (c) to produce monoclonal antibodies; and (e)screening the antibodies to select only those antibodies which bind todiseased and/or cancerous cells or cell lines but do not bind tonon-cancerous or normal cells or cell lines, or bind to normal cells ata lower level or in a different fashion.

In another aspect, the invention is a method of generating an anti-EphA2family antibody comprising culturing a host cell encoding such antibodyor progeny thereof under conditions that allow production of theantibody, and purifying the anti-EphA2 antibody.

In another aspect, the invention is a method of generating an anti-EphA2family antibody comprising culturing a host cell or progeny thereofunder conditions that allow production of the antibody, and purifyingthe anti-EPHA2 antibody.

In another aspect, the invention provides methods of generating any ofthe antibodies (or polypeptides) described herein by expressing one ormore polynucleotides encoding the antibody (which may be separatelyexpressed as a single light or heavy chain, or both a light and a heavychain are expressed from one vector) in a suitable cell, generallyfollowed by recovering and/or isolating the antibody or polypeptides ofinterest.

In another aspect, the invention is an anti-EphA2 antibody or apolypeptide (which may or may not be an antibody) that competitivelyinhibits preferential binding of an anti-EphA2 antibody to EphA2. Insome embodiments, the invention is an antibody or a polypeptide (whichmay or may not be an antibody) that binds preferentially to the same ordifferent epitopes on EphA2 as other anti-EphA2 antibodies.

In yet another aspect, the invention is a composition comprising EphA2bound by an antibody specific for an epitope of EphA2. In oneembodiment, the antibody is anti-EphA2. In other embodiments, two ormore anti-EphA2 antibodies are administered, with such antibodiesmapping to two or more different epitopes of EphA2. In some embodiments,the anti-EphA2 antibody is linked to a therapeutic agent or a detectablelabel.

In another aspect, the invention is an antibody comprising a fragment ora region of an anti-EphA2 antibody. In one embodiment, the fragment is alight chain of the antibody. In another embodiment, the fragment is aheavy chain of the antibody. In yet another embodiment, the fragmentcontains one or more variable regions from a light chain and/or a heavychain of the antibody. In yet another embodiment, the fragment containsone or more complementarity determining regions (CDRs) from a lightchain and/or a heavy chain of the antibody.

In another aspect, the invention provides polypeptides (which may or maynot be antibodies) comprising any of the following: a) one or more CDRs(or fragments thereof) from the light or heavy chain; b) three CDRs fromthe light chain; c) three CDRs from the heavy chain; d) three CDRs fromthe light chain and three CDRs from the heavy chain; e) the light chainvariable region; f) the heavy chain variable region of the anti-EphA2antibody.

In another aspect, the invention is a humanized antibody. In someembodiments, the humanized antibody comprises one or more CDRs of anon-human anti-EphA2 antibody. In some embodiments, the humanizedantibody binds to the same or different epitope(s) as other anti-EphA2antibodies. Generally, a humanized antibody of the invention comprisesone or more (one, two, three, four, five, six, or fragments thereof)CDRs which are the same and/or derived from the CDR(s) of the originalnon-human anti-EphA2 antibody. In some embodiments, the human antibodybinds to the same or different epitope(s) as other anti-EphA2antibodies. In anther aspect, the invention is a chimeric antibodycomprising variable regions derived from variable regions of a heavychain and a light chain of a non-human anti-EphA2 antibody and constantregions derived from constant regions of a heavy chain and a light chainof a human antibody.

In another aspect, the invention is an isolated polynucleotide thatencodes any one of antibodies LUCA19, LUCA40, SPL1 or SG5 that isproduced by a host cell with a deposit number of ATCC No. PTA-5070,PTA-6056, or PTA-6059, respectively, and host cell SG.3.15.B4.1F9 (ATCCNo. PTA-7356) or progeny thereof. This invention encompasses antibodypolypeptides having the inherent binding or biological activities of anyof the above-specified antibodies. In another aspect, the inventionprovides polynucleotides encoding any of the antibodies (includingantibody fragments) as well as any other polypeptides described herein.

In another aspect, the invention is a pharmaceutical compositioncomprising any of the polypeptides (including any of the antibodiesdescribed herein) or polynucleotides described herein, such aspharmaceutical compositions comprising an anti-EphA2 antibody linked toa chemotherapeutic agent, an antibody comprising a fragment of ananti-EphA2 antibody, a humanized antibody of a non-human anti-EphA2antibody, a chimeric antibody comprising variable regions derived fromvariable regions of a non-human anti-EphA2 antibody and constant regionsderived from constant regions of a human antibody, or a human antibodywith one or more properties of a non-human anti-EphA2 antibody, or anyof the anti-EphA2 antibody described herein linked to a chemotherapeuticagent (such as a radioactive moiety), and a pharmaceutically acceptableexcipient.

In one aspect, the invention is a composition comprising an anti-EphA2antibody bound to EphA2 present on a diseased or cancerous cell. Inpreferred embodiments, the cancer cell is selected from the groupconsisting of ovarian, lung, prostate, pancreatic, colon, and breastcancer cells. In some embodiments, the cancer cell is isolated. In someembodiments, the cancer cell is in a biological sample. Generally, thebiological sample is from an individual, such as a human.

In another aspect, the invention is a method of diagnosing disease in anindividual by detecting EphA2 on cells from the individual, particularlydiseases or disorders associated with inflammatory or autoimmuneresponses in individuals. In other aspects of the invention, methods areprovided for modulating inflammatory or autoimmune responses inindividuals. Diseases and conditions resulting from inflammation andautoimmune disorders that may be subject to treatment using thecompositions and methods of the invention include, by way ofillustration and not of limitation, multiple sclerosis, meningitis,encephalitis, stroke, other cerebral traumas, inflammatory bowel diseaseincluding ulcerative colitis and Crohn's disease, myasthenia gravis,lupus, rheumatoid arthritis, asthma, acute juvenile onset diabetes, AIDSdementia, atherosclerosis, nephritis, retinitis, atopic dermatitis,psoriasis, myocardial ischemia and acute leukocyte-mediated lung injury.

Still other indications for therapeutic use of antibodies and othertherapeutic agents of the invention include administration toindividuals at risk of organ or graft rejection. Over recent years therehas been a considerable improvement in the efficiency of surgicaltechniques for transplanting tissues and organs such as skin, kidney,liver, heart, lung, pancreas and bone marrow. Perhaps the principaloutstanding problem is the lack of satisfactory agents for inducingimmunotolerance in the recipient to the transplanted allograft or organ.When allogeneic cells or organs are transplanted into a host (i.e., thedonor and donee are different individuals from the same species), thehost immune system is likely to mount an immune response to foreignantigens in the transplant (host-versus-graft disease) leading todestruction of the transplanted tissue.

In another aspect, the invention is a method for diagnosing whether anindividual has cancer, comprising determining whether there isexpression of EphA2 on selected cells from the individual, wherein theexpression of EphA2 on said cells is indicative of said cancer. In someembodiments, the expression of EphA2 is determined using an anti-EphA2antibody. In some embodiments, the method involves detecting the levelof EphA2 expression from cells. The term “detection” as used hereinincludes qualitative and/or quantitative detection (measuring levels)with or without reference to a control.

In yet another aspect, the invention is a method of diagnosing cancer inan individual by detecting EphA2 on or released from cells from theindividual, wherein the cancer is selected from the group including butnot limited to adrenal gland tumors, AIDS-associated cancers, alveolarsoft part sarcoma, astrocytic tumors, bladder cancer (squamous cellcarcinoma and transitional cell carcinoma), bone cancer (adamantinoma,aneurismal bone cysts, osteochondroma, osteosarcoma), brain and spinalcord cancers, metastatic brain tumors, breast cancer, carotid bodytumors, cervical cancer, chondrosarcoma, dhordoma, chromophobe renalcell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer,cutaneous benign fibrous histiocytomas, desmoplastic small round celltumors, ependymomas, Ewing's tumors, extraskeletal myxoidchondrosarcoma, fibrogenesis imperfecta ossium, fibrous dysplasia of thebone, gallbladder and bile duct cancers, gestational trophoblasticdisease, germ cell tumors, head and neck cancers, islet cell tumors,Kaposi's Sarcoma, kidney cancer (nephroblastoma, papillary renal cellcarcinoma), leukemias, lipoma/benign lipomatous tumors,liposarcoma/malignant lipomatous tumors, liver cancer (hepatoblastoma,hepatocellular carcinoma), lymphomas, lung cancers (small cellcarcinoma, adenocarcinoma, squamous cell carcinoma, large cellcarcinoma, etc.), medulloblastoma, melanoma, meningiomas, multipleendocrine neoplasia, multiple myeloma, myelodysplastic syndrome,neuroblastoma, neuroendocrine tumors, ovarian cancer, pancreaticcancers, papillary thyroid carcinomas, parathyroid tumors, pediatriccancers, peripheral nerve sheath tumors, phaeochromocytoma, pituitarytumors, prostate cancer, posterious unveal melanoma, rare hematologicdisorders, renal metastatic cancer, rhabdoid tumor, rhabdomysarcoma,sarcomas, skin cancer, soft-tissue sarcomas, squamous cell cancer,stomach cancer, synovial sarcoma, testicular cancer, thymic carcinoma,thymoma, thyroid metastatic cancer, and uterine cancers (carcinoma ofthe cervix, endometrial carcinoma, and leiomyoma).

In another aspect, the invention is a method for aiding diagnosis ofcancer (such as but not limited to ovarian, lung, prostate, pancreatic,colon, or breast cancer) in an individual comprising determining theexpression of EphA2 in a biological sample from the individual. In someembodiments, the expression of EphA2 is determined using an anti-EphA2antibody. In some embodiments, the anti-EphA2 antibody is a familymember specifically named herein. In some embodiments, the method isdetecting the level of EphA2 expression from cells.

In yet another aspect, the invention is a method of treating cancer byadministering an effective amount of an antibody that binds to EphA2sufficient to reduce growth of cancerous cells. In some embodiments, theantibody is an anti-EphA2 antibody. In certain embodiments, thecancerous cells are selected from the group including but not limited toadrenal gland tumors, AIDS-associated cancers, alveolar soft partsarcoma, astrocytic tumors, bladder cancer (squamous cell carcinoma andtransitional cell carcinoma), bone cancer (adamantinoma, aneurismal bonecysts, osteochondroma, osteosarcoma), brain and spinal cord cancers,metastatic brain tumors, breast cancer, carotid body tumors, cervicalcancer, chondrosarcoma, dhordoma, chromophobe renal cell carcinoma,clear cell carcinoma, colon cancer, colorectal cancer, cutaneous benignfibrous histiocytomas, desmoplastic small round cell tumors,ependymomas, Ewing's tumors, extraskeletal myxoid chondrosarcoma,fibrogenesis imperfecta ossium, fibrous dysplasia of the bone,gallbladder and bile duct cancers, gestational trophoblastic disease,germ cell tumors, head and neck cancers, islet cell tumors, Kaposi'sSarcoma, kidney cancer (nephroblastoma, papillary renal cell carcinoma),leukemias, lipoma/benign lipomatous tumors, liposarcoma/malignantlipomatous tumors, liver cancer (hepatoblastoma, hepatocellularcarcinoma), lymphomas, lung cancers (small cell carcinoma,adenocarcinoma, squamous cell carcinoma, large carcinoma, etc.),medulloblastoma, melanoma, meningiomas, multiple endocrine neoplasia,multiple myeloma, myelodysplastic syndrome, neuroblastoma,neuroendocrine tumors, ovarian cancer, pancreatic cancers, papillarythyroid carcinomas, parathyroid tumors, pediatric cancers, peripheralnerve sheath tumors, phaeochromocytoma, pituitary tumors, prostatecancer, posterious unveal melanoma, rare hematologic disorders, renalmetastatic cancer, rhabdoid tumor, rhabdomysarcoma, sarcomas, skincancer, soft-tissue sarcomas, squamous cell cancer, stomach cancer,synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, thyroidmetastatic cancer, and uterine cancers (carcinoma of the cervix,endometrial carcinoma, and leiomyoma). In certain preferred embodiments,the cancerous cells are selected from the group of solid tumorsincluding but not limited to breast cancer, colon cancer, prostatecancer, lung cancer, sarcoma, renal metastatic cancer, thyroidmetastatic cancer, and clear cell carcinoma.

In yet another aspect, the invention is a method of delaying developmentof metastasis in an individual having cancer comprising administering aneffective amount of at least one of a family of antibodies that bindspecifically to EphA2. In one embodiment, the antibody is an anti-EphA2antibody. In another aspect, the invention is a method of inhibitinggrowth and/or proliferation of cancer cells in vitro or in an individualcomprising administering an effective amount of a composition comprisingan anti-EphA2 antibody associated with (including linked to) achemotherapeutic agent to the cell culture or sample, or to theindividual.

In yet another aspect, the invention is a method of delivering atherapeutic agent to a cancerous cell in an individual by administeringto the individual an effective amount of at least one member of a familyof antibodies, which bind specifically to EphA2. In other embodiments,an anti-EphA2 antibody is delivered to an individual in combination with(including linked to) another therapeutic agent.

In some embodiments, the anti-EphA2 antibody is a humanized antibodyderived from a named antibody family member herein (generally, but notnecessarily, comprising one or more partial or intact CDRs of theantibody). In some embodiments, the anti-EphA2 antibody is a humanantibody with one or more properties of the named antibody familymember. In some embodiments, the chemotherapeutic agent (such as a toxinor a radioactive molecule) is delivered into the cancer cells (isinternalized). In some embodiments, the agent is saporin.

In another aspect, the invention is a method of treating cancer in anindividual comprising administering an effective amount of a compositioncomprising an anti-EphA2 antibody associated with (including linked to)a chemotherapeutic agent to the individual.

The present invention further provides methods for modulating, either byenhancing or reducing, the association of EphA2 with a cytoplasmicsignaling partner. The association of EphA2 with a cytoplasmic signalingpartner can be impacted by contacting an EphA2 molecule presenting on acell surface, with an agent that modulates the binding of the signalingpartner to EphA2. Agents which block or reduce EphA2 association withits binding and/or signaling partners can be used to modulate biologicaland pathological processes which are involved in EphA2 -mediatedinflammation or immune responses. Pathological processes involving thisaction include tumor-associated cell growth.

Agents can be tested for their ability to block, reduce, enhance orotherwise modulate the association of EphA2 with a binding partner, suchas an anti-EphA2 antibody. Specifically, an agent can be tested for theability to modulate such an interaction by incubating a peptidecomprising the EphA2 interaction site (typically in its nativeconformation as it exists on intact living cells) with a binding partnerand a test agent, and determining whether the test agent reduces orenhances the binding of the binding partner to the EphA2 peptide.Agonists, antagonists, and other modulators are expressly contemplated.

In certain aspects, the invention is a method for aiding in thediagnosis of disease in an individual comprising the steps of (i)assaying for the presence of EphA2 in a blood or tissue sample obtainedfrom an individual; (ii) detecting whether said sample has an increasedamount of a EphA2 marker relative to a normal (non-diseased) blood ortissue sample; and (iii) correlating an increased amount of said markerto a positive diagnosis or correlating the absence of an increasedamount of said marker to a negative diagnosis for disease. In certainembodiments, the marker is detected using an anti-EphA2 antibody. Incertain embodiments, the method is effected by a technique selected fromthe group consisting of radionuclide imaging, flow cytometry, andimmunohistochemistry.

In another aspect, the invention is a method of diagnosing cancer ormetastatic cancer in an individual by detecting EphA2 on cells from theindividual using the anti-EphA2 antibody or any EphA2 binding moiety(polypeptides, including, but not limited to, various antibodies andantibody derivatives) described herein. In some embodiments, the canceris melanomas, breast, colon, lung, and prostate. In some embodiments,the method is detecting the level of EphA2 from cells. The presence ofEphA2 is detected by detecting a complex between EphA2 and an EphA2binding moiety. The term “detection” as used herein includes qualitativeand/or quantitative detection (measuring levels) with or withoutreference to a control.

In another aspect, the invention is a method of treating cancer byadministering an effective amount of a composition comprising theanti-EphA2 antibody, or any of the antibodies (including polypeptides)or polynucleotides embodiments described herein, including but notlimited to the anti-EphA2 antibody associated with a therapeutic agent,an antibody comprising a fragment or a region of the anti-EphA2antibody, a humanized antibody (generally, but not necessarily,comprising one or more CDRs of the anti-EphA2 antibody), a chimericantibody comprising variable regions derived from variable regions ofthe anti-EphA2 antibody and constant regions derived from constantregions of a human antibody, or a human antibody with one or moreproperties of the anti-EphA2 antibody, sufficient to reduce growth ofcancerous cells. In some embodiments, the cancer is melanomas, breast,colon, lung, or prostate.

In certain embodiments, the invention is a method of treating cancer ina patient, said method comprising administering to said patient atherapeutically effective amount of an EphA2 antibody that specificallybinds to EphA2, and has at least one, and preferably two or more, of thefollowing characteristics of SPL1, LUCA19, SG5 or LUCA40:

-   -   a. the ability to bind to EphA2 on a cancer cell;    -   b. the ability to bind to a portion of EphA2 that is exposed on        the surface of a living cancer cell in vitro or in vivo;    -   c. the ability to impede proliferation of tumor cells without        increasing the phosphotyrosine content of EphA2;    -   d. the ability to deliver a therapeutic agent or detectable        marker to a cancer cell expressing EphA2; and    -   e. the ability to deliver a therapeutic agent or detectable        marker into a cancer cell expressing EphA2.

In another aspect, the invention is a method of inhibiting growth and/orproliferation of cancerous cells in an individual by administering tothe individual an effective amount of a composition comprising theanti-EphA2 antibody, or any of the antibodies (including polypeptides)or polynucleotides embodiments described herein, including but notlimited to the anti-EphA2 antibody associated with a therapeutic agent,an antibody comprising a fragment or a region of the anti-EphA2antibody, a humanized antibody (generally, but not necessarily,comprising one or more CDRs of the anti-EphA2 antibody), a chimericantibody comprising variable regions derived from variable regions ofthe anti-EphA2 antibody and constant regions derived from constantregions of a human antibody, or a human antibody with one or moreproperties of the anti-EphA2 antibody, sufficient to reduce growth ofcancerous cells. In some embodiments, the cancer is melanomas, breast,colon, lung, or prostate.

In another aspect, the invention is a method of preventing or delayingdevelopment of metastasis, treating metastatic cancer, or inhibitingproliferation of metastatic cancer cells in an individual with cancer byadministering an effective amount of a composition comprising theanti-EphA2 antibody, or any of the antibodies (including polypeptides)or polynucleotides embodiments described herein, including but notlimited to the anti-EphA2 antibody associated with a therapeutic agent,an antibody comprising a fragment or a region of the anti-EphA2antibody, or a humanized antibody (generally, but not necessarily,comprising one or more CDRs of the anti-EphA2 antibody), a chimericantibody comprising variable regions derived from variable regions ofthe anti-EphA2 antibody and constant regions derived from constantregions of a human antibody, or a human antibody with one or moreproperties of the anti-EphA2 antibody, sufficient to reduce growth ofcancerous cells. In some embodiments, the cancer is melanomas, breast,colon, lung, or prostate.

In another aspect, the invention is a method of delivering a therapeuticagent (such as a toxin, or a radioactive molecule) to cancerous cells inan individual by administering to the individual an effective amount ofan EphA2 binding antibody or any EphA2 binding moiety (polypeptides,including but not limited to antibodies or antibody derivatives)described herein that are linked to a therapeutic agent (such as a toxinor a radioactive molecule). The EphA2 binding moiety includes, but notlimited to the anti-EphA2 antibody, an antibody comprising a fragment ora region of the anti-EphA2 antibody, or a humanized antibody (generally,but not necessarily, comprising one or more CDRs of the anti-EphA2antibody), a chimeric antibody comprising variable regions derived fromvariable regions of the anti-EphA2 antibody and constant regions derivedfrom constant regions of a human antibody, or a human antibody with oneor more properties of the anti-EphA2 antibody. In some embodiments, thecancerous cells are from melanomas, breast, colon, lung, or prostatecancer. In another embodiment, the therapeutic agent (such as a toxin ora radioactive molecule) is delivered into the cancerous cells (isinternalized). Accordingly, the invention provides methods of inhibitinggrowth and/or proliferation of cancer cells such that the therapeuticagent is delivered into those cancer cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows results of A549 tumor xenografts subcutaneously treatedwith SPL1 and their slow re-growth after cessation of dosing.

DETAILED DESCRIPTION OF THE INVENTION

The invention disclosed herein provides antibodies and polypeptideswhich bind to an antigen, EphA2 and methods of making and using theseantibodies and polypeptides to diagnose and treat various diseases humancancers associated with expression and/or overexpression of EphA2. EphA2has been shown to be present and its expression is increased in avariety of human cancers. Anti-EphA2 antibodies such as those producedby any one of the host cells identified in the following paragraph havebeen generated and have been shown to specifically bind to EphA2.

In accordance with the Budapest Treaty, the hybridoma which producesmLUCA19 has been deposited in the American Type Culture Collection(ATCC) 10801 University Blvd., Manassas Va. 20110-2209 on Mar. 20, 2003with a Patent Deposit Designation of PTA-5070, the hybridoma whichproduces SPL1 has been deposited in the American Type Culture Collection(ATCC) on Jun. 8, 2004 with a Patent Deposit Designation of PTA-6059,the hybridoma which produces LUCA40 has been deposited in the AmericanType Culture Collection (ATCC) on Jun. 8, 2004 with a Patent DepositDesignation of PTA-6056, and the hybridoma which produces SG5 has beendeposited in the American Type Culture Collection (ATCC) on Feb. 2, 2006with a Patent Deposit Designation of PTA-7356.

I. General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, Molecular Cloning: ALaboratory Manual, second edition (Sambrook et al., 1989) Cold SpringHarbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methodsin Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook(J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I.Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P.Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture:Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell,eds., 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press,Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C.Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.Miller and M. P. Calos, eds., 1987); Current Protocols in MolecularBiology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase ChainReaction, (Mullis et al., eds., 1994); Current Protocols in Immunology(J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology(Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers,1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D.Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practicalapproach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000);Using antibodies: a laboratory manual (E. Harlow and D. Lane (ColdSpring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer:Principles and Practice of Oncology (V. T. DeVita et al., eds., J.B.Lippincott Company, 1993).

II. Definitions

EphA2 refers to that polypeptide antigen with a molecular weight ofapproximately 130 kD, against which the antibodies of the presentinvention are directed. As described in more detail herein, this antigenhas more than one different epitope. Some of the preferred antibodyembodiments of this invention are directed against one of two or morespecific epitopes of the EphA2 antigen. It is currently believed thatEphA2 is over-expressed in certain cancer cells in comparison to theirnormal tissue counterparts

An “antibody” is an immunoglobulin molecule capable of specific bindingto a target, such as a carbohydrate, polynucleotide, lipid, polypeptide,etc., through at least one antigen recognition site, located in thevariable region of the immunoglobulin molecule. As used herein, the termencompasses not only intact polyclonal or monoclonal antibodies, butalso fragments thereof (such as Fab, Fab′, F(ab′)₂, Fv), single chain(ScFv), mutants thereof, naturally occurring variants, fusion proteinscomprising an antibody portion with an antigen recognition site of therequired specificity, humanized antibodies, chimeric antibodies, and anyother modified configuration of the immunoglobulin molecule thatcomprises an antigen recognition site of the required specificity.

The term “monoclonal antibody” encompasses not only intact monoclonalantibodies and full-length monoclonal antibodies, but also fragmentsthereof (such as Fab, Fab′, F(ab′)₂, Fv), single chain (ScFv), mutantsthereof, fusion proteins comprising an antibody portion, humanizedmonoclonal antibodies, chimeric monoclonal antibodies, and any othermodified configuration of the immunoglobulin molecule that comprises anantigen recognition site of the required specificity and the ability tobind to an antigen. It is not intended to be limited as regards to thesource of the antibody or the manner in which it is made (e.g., byhybridoma, phage selection, recombinant expression, transgenic animals,etc.).

“Humanized” antibodies refer to a molecule having an antigen-bindingsite that is substantially derived from an immunoglobulin from anon-human species and the remaining immunoglobulin structure of themolecule based upon the structure and/or sequence of a humanimmunoglobulin. The antigen-binding site may comprise either completevariable domains fused onto constant domains or only the complementaritydetermining regions (CDRs) grafted onto appropriate framework regions inthe variable domains. Antigen binding sites may be wild type or modifiedby one or more amino acid substitutions, e.g., modified to resemblehuman immunoglobulin more closely. Some forms of humanized antibodiespreserve all CDR sequences (for example, a humanized mouse antibodywhich contains all six CDRs from the mouse antibodies). Other forms ofhumanized antibodies have one or more CDRs (one, two, three, four, five,six) which are altered with respect to the original antibody, which arealso termed one or more CDRs “derived from” one or more CDRs fromLUCA19, SPL1, SG5 or LUCA40.

“Chimeric antibodies” refers to those antibodies wherein one portion ofeach of the amino acid sequences of heavy and light chains is homologousto corresponding sequences in antibodies derived from a particularspecies or belonging to a particular class, while the remaining segmentof the chains is homologous to corresponding sequences in another.Typically, in these chimeric antibodies, the variable region of bothlight and heavy chains mimics the variable regions of antibodies derivedfrom one species of mammals, while the constant portions are homologousto the sequences in antibodies derived from another. One clear advantageto such chimeric forms is that, for example, the variable regions canconveniently be derived from presently known sources using readilyavailable hybridomas or B cells from non human host organisms incombination with constant regions derived from, for example, human cellpreparations. While the variable region has the advantage of ease ofpreparation, and the specificity is not affected by its source, theconstant region being human, is less likely to elicit an immune responsefrom a human subject when the antibodies are injected than would theconstant region from a non-human source. However, the definition is notlimited to this particular example.

An epitope that “specifically binds” or “preferentially binds” (usedinterchangeably herein) to an antibody or a polypeptide is a term wellunderstood in the art, and methods to determine such specific orpreferential binding are also well known in the art. A molecule is saidto exhibit “specific binding” or “preferential binding” if it reacts orassociates more frequently, more rapidly, with greater duration and/orwith greater affinity with a particular cell or substance than it doeswith alternative cells or substances. An antibody “specifically binds”or “preferentially binds” to a target if it binds with greater affinity,avidity, more readily, and/or with greater duration than it binds toother substances. For example, an antibody that specifically orpreferentially binds to an EphA2 epitope is an antibody that binds thisEphA2 epitope with greater affinity, avidity, more readily, and/or withgreater duration than it binds to other EphA2 epitopes or non-EphA2epitopes. It is also understood by reading this definition that, forexample, an antibody (or moiety or epitope) that specifically orpreferentially binds to a first target may or may not specifically orpreferentially bind to a second target. As such, “specific binding” or“preferential binding” does not necessarily require (although it caninclude) exclusive binding. Generally, but not necessarily, reference tobinding means preferential binding.

The term “immunologically active” in reference to an epitope being or“remaining immunologically active” refers to the ability of an antibody(e.g., anti-EphA2 antibody) to bind to the epitope under differentconditions, for example, after the epitope has been subjected toreducing and denaturing conditions.

As used herein, the terms “LUCA19, SPL1, LUCA40, or SG5”, “anti-EphA2antibody” and “monoclonal anti-EphA2 antibody” are used interchangeablyto refer to immunoglobulin produced by any of the host cells with adeposit number of ATCC No. PTA-5070, PTA-6056, or PTA-6059, or host cellSG.3.15B4.1F9 (ATCC No. PTA-7356) or progeny thereof. Differentbiological functions are associated with LUCA19, SPL1, SG5 or LUCA40,including, but not limited to, ability to bind to EphA2; ability to bindto EphA2 extracellular domain; ability to bind to EphA2 on cancer cellsexposed on the surface of a living cell in vitro or in vivo; ability todeliver a chemotherapeutic agent to cancerous cells (such as ovarian,prostate, pancreatic, lung, colon, or breast cancer cells) expressingEphA2; ability to deliver a therapeutic agent or detectable marker intocancer cells expressing EphA2. As discussed herein, polypeptides(including antibodies) of the invention may have any one or more ofthese characteristics.

An “anti-EphA2 equivalent antibody” or “anti-EphA2 equivalentpolypeptide” refers to an antibody or a polypeptide having one or morebiological functions associated with an anti-EphA2 antibody, such as,for example binding specificity.

As used herein, “agent” refers to a biological, pharmaceutical, orchemical compound. Non-limiting examples include simple or complexorganic or inorganic molecule, a peptide, a protein, an oligonucleotide,an antibody, an antibody derivative, antibody fragment, a vitaminderivative, a carbohydrate, a toxin, or a chemotherapeutic compound.Various compounds can be synthesized, for example, small molecules andoligomers (e.g., oligopeptides and oligonucleotides), and syntheticorganic compounds based on various core structures. In addition, variousnatural sources can provide compounds for screening, such as plant oranimal extracts, and the like. A skilled artisan can readily recognizethat there is no limit as to the structural nature of the agents of thepresent invention.

Agents that are employed in the methods of this invention can berandomly selected or rationally selected or designed. As used herein, anagent is said to be randomly selected when the agent is chosen randomlywithout considering the specific sequences involved in the associationof EphA2 with its native binding partners or known antibodies. Anexample of randomly selected agents is the use of a chemical library ora peptide combinatorial library.

As used herein, an agent is said to be rationally selected or designedwhen the agent is chosen on a nonrandom basis that takes into accountthe sequence of the target site and/or its conformation in connectionwith the agent's action. With respect to anti-EphA2 agents, it iscurrently believed that there are at least three epitopes on EphA2against which antibodies can be raised and therefore at least threesites of action for agents that block EphA2/anti-EphA2 interaction. Thisinvention also encompasses agents that act at the sites of interactionbetween EphA2 and its native binding partner, although other ligands andtheir active EphA2 -interactive sites are also encompassed within thescope of this invention, whether currently known or later identified.Agents can be rationally selected or rationally designed by utilizingthe peptide sequences that make up the contact sites of thereceptor/ligand and/or EphA2/anti-EphA2 antibody complex. For example, arationally selected peptide agent can be a peptide whose amino acidsequence is identical to an epitope appearing on EphA2 as it is exposedon the surface of a living cell in its native environment. Such an agentwill reduce or block the association of the anti-EphA2 antibody withEphA2, or the association of EphA2 with its native ligand, as desired,by binding to the anti-EphA2 antibody or to the native ligand.

As used herein, the term “labeled”, with regard to the antibody, isintended to encompass direct labeling of the antibody by coupling (i.e.,physically linking) a detectable substance, such as a radioactive agentor a fluorophore (e.g. fluorescein isothiocyanate (FITC) orphycoerythrin (PE)) to the antibody, as well as indirect labeling of theprobe or antibody by reactivity with a detectable substance.

As used herein, the term “association”, with regard to the antibody,includes covalent and non-covalent attachment or binding to an agent(e.g., chemotherapeutic agent). The antibody can be associated with anagent (e.g., chemotherapeutic agent) by direct binding or indirectbinding via attachment to a common platform, such that the antibodydirects the localization of the agent to the cancerous cell to which theantibody binds and wherein the antibody and agent do not substantiallydissociate under physiological conditions such that the agent is nottargeted to the same cancerous cell to which the antibody binds or suchthat the agent's potency is not decreased.

A “biological sample” encompasses a variety of sample types obtainedfrom an individual and can be used in a diagnostic or monitoring assay.The definition encompasses blood and other liquid samples of biologicalorigin, solid tissue samples such as a biopsy specimen or tissuecultures or cells derived therefrom, and the progeny thereof, forexample, cells obtained from a tissue sample collected from anindividual suspected of having cancer, in preferred embodiments fromovary, lung, prostate, pancreas, colon, and breast tissue. Thedefinition also includes samples that have been manipulated in any wayafter their procurement, such as by treatment with reagents,solubilization, or enrichment for certain components, such as proteinsor polynucleotides, or embedding in a semi-solid or solid matrix forsectioning purposes. The term “biological sample” encompasses a clinicalsample, and also includes cells in culture, cell supernatants, celllysates, serum, plasma, biological fluid, and tissue samples.

The terms “polypeptide”, “oligopeptide”, “peptide” and “protein” areused interchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified naturally orby intervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Alsoincluded within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids, etc.), as well as other modifications known in the art. Itis understood that, because the polypeptides of this invention are basedupon an antibody, the polypeptides can occur as single chains orassociated chains.

A “variable region” of an antibody refers to the variable region of theantibody light chain or the variable region of the antibody heavy chain,either alone or in combination.

A “constant region” of an antibody refers to the constant region of theantibody light chain or the constant region of the antibody heavy chain,either alone or in combination.

As used herein, “substantially pure” refers to material that is at least50% pure (i.e., free from contaminants), more preferably at least 90%pure, more preferably at least 95% pure, more preferably at least 98%pure, more preferably at least 99% pure.

A “host cell” includes an individual cell or cell culture that can be orhas been a recipient for vector(s) for incorporation of polynucleotideinserts. Host cells include progeny of a single host cell, and theprogeny may not necessarily be completely identical (in morphology or ingenomic DNA complement) to the original parent cell due to natural,accidental, or deliberate mutation. A host cell includes cellstransfected in vivo with a polynucleotide(s) of this invention.

As used herein, “delaying development of metastasis” means to defer,hinder, slow, retard, stabilize, and/or postpone development ofmetastasis. This delay can be of varying lengths of time, depending onthe history of the cancer and individual being treated. As is evident toone skilled in the art, a sufficient or significant delay can, ineffect, encompass prevention, in that the individual does not developthe metastasis.

An “effective amount” of a drug, compound, or pharmaceutical compositionis an amount sufficient to effect beneficial or desired resultsincluding, without limitation, clinical results such as shrinking thesize of the tumor (in the cancer context, for example, breast orprostate cancer), retardation of cancerous cell growth, delaying thedevelopment of metastasis, decreasing one or more symptoms resultingfrom the disease, increasing the quality of life of those suffering fromthe disease, decreasing the dose of other medications required to treatthe disease, enhancing the effect of another medication such as viatargeting and/or internalization, delaying the progression of thedisease, and/or prolonging survival of individuals. An effective amountcan be administered in one or more administrations. For purposes of thisinvention, an effective amount of drug, compound, or pharmaceuticalcomposition is an amount sufficient to reduce the proliferation of (ordestroy) neoplastic cells and/or to reduce and/or delay the development,or growth, of metastases of neoplastic cells, either directly orindirectly.

As is understood in the cancer clinical context, an effective amount ofa drug, compound, or pharmaceutical composition may or may not beachieved in conjunction with another drug, compound, or pharmaceuticalcomposition. Thus, an “effective amount” may be considered in thecontext of administering one or more therapeutic agents, and a singleagent may be considered to be given in an effective amount if, inconjunction with one or more other agents, a desirable result may be oris achieved.

As used herein, “treatment” or “treating” is an approach for obtainingbeneficial or desired results including preferably clinical results. Forpurposes of this invention, beneficial or desired clinical resultsinclude, but are not limited to, one or more of the following: reducingthe proliferation of (or destroying) neoplastic cells, reducingmetastasis of neoplastic cells found in cancers, shrinking the size ofthe tumor, decreasing symptoms resulting from the disease, increasingthe quality of life of those suffering from the disease, decreasing thedose of other medications required to treat the disease, delaying theprogression of the disease, and/or prolonging survival of patients.

As used herein, “delaying development of metastasis” means to defer,hinder, slow, retard, stabilize, and/or postpone development ofmetastasis. This delay can be of varying lengths of time, depending onthe history of the cancer and/or individual being treated. As is evidentto one skilled in the art, a sufficient or significant delay can, ineffect, encompass prevention, in that the individual does not developthe metastasis.

A “biological sample” encompasses a variety of sample types obtainedfrom an individual and can be used in a diagnostic or monitoring assay.The definition encompasses blood and other liquid samples of biologicalorigin, solid tissue samples such as a biopsy specimen or tissuecultures or cells derived therefrom, and the progeny thereof. Thedefinition also includes samples that have been manipulated in any wayafter their procurement, such as by treatment with reagents,solubilization, or enrichment for certain components, such as proteinsor polynucleotides, or embedding in a semi-solid or solid matrix forsectioning purposes. The term “biological sample” encompasses a clinicalsample, and also includes cells in culture, cell supernatants, celllysates, serum, plasma, biological fluid, and tissue samples.

An “individual” is a vertebrate, preferably a mammal, more preferably ahuman. Mammals include, but are not limited to, farm animals, sportanimals, pets (such as cats, dogs, horses), primates, mice and rats.

“Toxin” or “cytotoxin” refers to any substance, which effects an adverseresponse within a cell. For example, a toxin directed to a cancerouscell would have an adverse, sometimes deleterious effect, on thecancerous cell.

As used herein, “agent” refers to a biological, pharmaceutical, orchemical compound. Non-limiting examples include simple or complexorganic or inorganic molecule, a peptide, a protein, an oligonucleotide,an antibody, an antibody derivative, or antibody fragment. Variouscompounds can be synthesized, for example, small molecules and oligomers(e.g., oligopeptides and oligonucleotides), and synthetic organiccompounds based on various core structures. In addition, various naturalsources can provide compounds for screening, such as plant or animalextracts, and the like.

As used herein, a “therapeutic agent” means any agent useful for therapy(here, generally in the cancer context) including anti-tumor drugs,toxins or cytotoxins, cytotoxin agents, and radioactive agents.

“Active immune response” refers to the development of, and on-goingproduction of, antibodies in vivo directed against an antigen, inresponse to the administration of the antigen, or DNA vectors coding forthat antigen, to the host mammal by intravenous, intramuscular,subcutaneous, or other mode of administration with or without anadjuvant. Active immune response can also include other aspects of theimmune response, such as a cellular immune response.

Compositions and Methods of Making the Compositions

This invention encompasses compositions, including pharmaceuticalcompositions, comprising antibodies, polypeptides and proteins that bindto EphA2, and polynucleotides comprising sequences encoding antibodies,polypeptides and proteins that bind to EphA2. As used herein,compositions comprise one or more antibodies, polypeptides and/orproteins that bind to EphA2, and/or one or more polynucleotidescomprising sequences encoding one or more antibodies, polypeptides andproteins that bind to EphA2. These compositions may further comprisesuitable excipients, such as pharmaceutically acceptable excipientsincluding buffers, which are well known in the art.

The present invention also encompasses various formulations of LUCA19,SPL1, SG5 or LUCA40 and equivalent antibodies or polypeptide fragments(e.g., Fab, Fab′, F(ab′)₂, Fv, Fc, etc.), chimeric antibodies, singlechain (ScFv), mutants thereof, fusion proteins comprising an antibodyportion, humanized antibodies, and any other modified configuration ofLUCA19, SPL1, SG5 or LUCA40 that comprises an antigen (EphA2),recognition site of the required specificity. The invention alsoprovides human antibodies displaying one or more of the biologicalcharacteristics of LUCA19, SPL1, SG5 or LUCA40. The equivalentantibodies of LUCA19, SPL1, SG5 or LUCA40 (including humanizedantibodies and human antibodies), polypeptide fragments of LUCA19, SPL1,SG5 or LUCA40, and polypeptides comprising any of these fragments areidentified and characterized by one or more characteristics of LUCA19,SPL1, SG5 or LUCA40.

In some embodiments, the antibodies, polypeptides and proteins of theinvention that bind to EphA2 are antibodies, polypeptides and proteinsthat competitively inhibit preferential binding of LUCA19, SPL1, SG5 orLUCA40 to EphA2 or that preferentially bind to the same epitope on EphA2as the anti-EphA2 antibody preferentially binds.

Accordingly, the invention provides any of the following (orcompositions, including pharmaceutical compositions), comprising any ofthe following: (a) anti-EphA2 antibody produced by the host cell with adeposit number of ATCC No. PTA-5070, PTA-6056, or PTA-6059, or host cellSG3.15B4.1F9 (PTA-7356) or its progeny; (b) a humanized form ofanti-EphA2 antibody; (c) an antibody comprising one or more of the lightchain and/or heavy chain variable regions of anti-EphA2 antibody; (d) achimeric antibody comprising variable regions homologous or derived fromvariable regions of a heavy chain and a light chain of anti-EphA2antibody, and constant regions homologous or derived from constantregions of a heavy chain and a light chain of a human antibody; (e) anantibody comprising one or more of the light chain and/or heavy chainCDRs (at least one, two, three, four, five, or six) of LUCA19, SPL1, SG5or LUCA40; (f) an antibody comprising a heavy and/or a light chain ofLUCA19, SPL1, SG5 or LUCA40; (g) a human antibody that is equivalent toLUCA19, SPL1, SG5 or LUCA40. A humanized form of the antibody may or maynot have CDRs identical to LUCA19, SPL1, LUCA40, or SG5 or antibodyproduced by the host cell with a deposit number of ATCC No. PTA-5070,PTA-6056, or PTA-6059 or host cell SG3.15B4.1F9 (ATCC No. PTA-7356).Determination of CDR regions is well within the skill of the art. Insome embodiments, the invention provides an antibody which comprises atleast one CDR that is substantially homologous to at least one CDR, atleast two, at least three, at least four, at least 5 CDRs of LUCA19,SPL1, SG5 or LUCA40 (or, in some embodiments substantially homologous toall 6 CDRs of LUCA19, SPL1, SG5 or LUCA40, or derived from LUCA19, SPL1,LUCA40, or SG5), or antibody produced by the host cell with a depositnumber of ATCC No. PTA-5070, PTA-6056, PTA-6059, or host cellSG3.15B4.1F9 (ATCC No. PTA-XXXX). Other embodiments include antibodiesthat have at least two, three, four, five, or six CDR(s) that aresubstantially homologous to at least two, three, four, five or six CDRsof LUCA19, SPL1 LUCA40, or SG5 or derived from LUCA19, SPL1, LUCA40, orSG5, or antibody produced by the host cell with a deposit number of ATCCNo. PTA-5070, PTA-6056, PTA-6059 or host cell SG3.15B.1F9. It isunderstood that, for purposes of this invention, binding specificity andoverall activity (which may be in terms of reducing the growth and/orproliferation of cancerous cells, inducing apoptotic cell death in thecancer cell, delaying the development of metastasis, and treatingpalliatively) is generally retained, although the extent of activity mayvary compared to LUCA19, SPL1, SG5 or LUCA40 (may be greater or lesser).The invention also provides methods of making any of these antibodies.Methods of making antibodies are known in the art and are describedherein.

The invention also provides polypeptides comprising an amino acidsequence of the antibodies of the invention, such as LUCA19, SPL1, SG5or LUCA40. In some embodiments, the polypeptide comprises one or more ofthe light chain and heavy chain variable regions of the anti-EphA2antibody. In some embodiments, the polypeptide comprises one or more ofthe light chain and heavy chain CDRs of LUCA19, SPL1, SG5 or LUCA40. Insome embodiments, the polypeptide comprises three CDRs of the lightchain and heavy chain of LUCA19, SPL1, SG5 or LUCA40. In someembodiments, the polypeptide comprises an amino acid sequence of LUCA19,SPL1, SG5 or LUCA40 that has any of the following: at least 5 contiguousamino acids of a sequence of LUCA19, SPL1, SG5 or LUCA40, at least 8contiguous amino acids, at least about 10 contiguous amino acids, atleast about 15 contiguous amino acids, at least about 20 contiguousamino acids, at least about 25 contiguous amino acids, at least about 30contiguous amino acids, wherein at least 3 of the amino acids are from avariable region of LUCA19, SPL1, SG5 or LUCA40. In one embodiment, thevariable region is from a light chain of LUCA19, SPL1, SG5 or LUCA40. Inanother embodiment, the variable region is from a heavy chain of LUCA19,SPL1, SG5 or LUCA40. In another embodiment, the 5 (or more) contiguousamino acids are from a complementarity-determining region (CDR) ofLUCA19, SPL1, SG5 or LUCA40.

Antibodies may be polyclonal (e.g., not homogeneous) or monoclonal.Methods of making monoclonal antibodies are known in the art. One methodwhich may be employed is the method of Kohler and Milstein, Nature256:495-497 (1975) or a modification thereof. In general, a mouse or ratis used for immunization but other animals may also be used. Theimmunogen can be, but is not limited to, primary cells, cultured celllines, cancerous cells, nucleic acids, tissue, or peptides. Full lengthEphA2 or any fragments of EphA2 (e.g., extracellular domain), or EphA2expressing cancer cells are used as immunogen. Cells used for immunogen,for example, EphA2 expressing cancer cells may be cultured for a periodof time (at least 24 hours) prior to their use as an immunogen. Cellsmay be used as immunogens by themselves or in combination with anon-denaturing adjuvant, such as Ribi. In general, cells should be keptintact and preferably viable when used as immunogen. Methods ofgenerating antibodies using intact cells as immunogen are described inWO 00/37503. Intact cells may allow antigens to be better detected thanruptured cells. In addition, monoclonal antibodies generated usingintact cells as immunogen are most likely against an antigenicdeterminant on the cell surface or against extracellular domain of atransmembrane receptor. Use of denaturing or harsh adjuvants, e.g.,Freud's adjuvant, may rupture the cells and therefore is discouraged.The immunogen may be administered multiple times at periodic intervalssuch as, bi-weekly, or weekly, or may be administered in such a way asto maintain viability in the animal (e.g., in a tissue recombinant).

To monitor the antibody response, a small biological sample (e.g.,blood) may be obtained from the animal and tested for antibody titeragainst the immunogen. The spleen and/or several large lymph nodes canbe removed and dissociated into single cells. If desired, the spleencells may be screened (after removal of non-specifically adherent cells)by applying a cell suspension to a plate or to a well coated with theantigen. B-cells, expressing membrane-bound immunoglobulin specific forthe antigen, will bind to the plate, and are not rinsed away with therest of the suspension. Resulting B-cells, or all dissociated spleencells, can then be fused with myeloma cells (e.g., X63-Ag8.653 and thosefrom the Salk Institute, Cell Distribution Center, San Diego, Calif.).Polyethylene glycol (PEG) may be used to fuse spleen or lymphocytes withmyeloma cells to form a hybridoma. The hybridoma is then cultured in aselective medium (e.g., hypoxanthine, aminopterin, thymidine medium,otherwise known as “HAT medium”). The resulting hybridomas are thenplated by limiting dilution, and are assayed for the production ofantibodies that bind specifically to the immunogen (e.g., surface ofcancer cell lines, EphA2, etc.) using FACS, immunohistochemistry (IHCscreening), and Western blot. The selected monoclonal antibody-secretinghybridomas are then cultured either in vitro (e.g., in tissue culturebottles or hollow fiber reactors), or in vivo (e.g., as ascites inmice). Methods of culturing hybridoma under conditions to generate theanti-EphA2 antibody, and purifying the antibody are known in the art.

Monoclonal antibody-secreting hybridomas described above can be selectedfor producing antibodies that bind preferentially to the epitope onEphA2 that the anti-EphA2 antibody preferentially binds. Methods ofselecting such antibody are known in the art. For example, bindingcompetition assays can be used to determine whether an antibody binds tothe same epitope as does LUCA19, SPL1, SG5 or LUCA40. An antibody'scompetition with LUCA19, SPL1, SG5 or LUCA40 for binding to EphA2indicates that the antibody binds preferentially to the epitope thatLUCA19, SPL1, SG5 or LUCA40 binds. Binding competition assays are wellknown in the art. Binding competition assays, which can be configured ina number of different formats, using either labeled antigen or labeledantibody. Usually, the antigen is immobilized on a 96-well plate, andthe ability of unlabeled antibodies to block the binding of labeledantibodies is measured using radioactive or enzyme labels. Polypeptidesthat bind preferentially to the epitope on EphA2 that the anti-EphA2antibody binds preferentially can also be tested and identified usingsimilar methods.

As another alternative to the cell fusion technique, EBV immortalized Bcells may be used to produce monoclonal antibodies of the subjectinvention. The hybridomas are expanded and subcloned, if desired, andsupernatants are assayed for anti-immunogen activity by conventionalassay procedures (e.g., FACS, IHC, radioimmunoassay, enzyme immunoassay,fluorescence immunoassay, etc.).

In another alternative, the antibodies can be made recombinantly.Methods for making recombinant antibodies are well known in the art.Monoclonal antibody LUCA19, SPL1, SG5 or LUCA40 and any other equivalentantibodies can be sequenced and produced recombinantly in vitro. In oneembodiment, LUCA19, SPL1, SG5 or LUCA40 is sequenced and thepolynucleotide sequence is then cloned into a vector for expression orpropagation. The sequence encoding the antibody of interest may bemaintained in a vector in a host cell and the host cell can then beexpanded and frozen for future use. In another alternative, antibodiesmay be made recombinantly by phage display technology. See, for example,U.S. Pat. Nos. 5,565,332; 5,580,717; 5,733,743; 6,265,150; and Winter etal., Annu. Rev. Immunol. (1994) 12:433-455.

In another alternative, the anti-EphA2 antibody or any other antibodiesor protein of interest may be subjected to sequencing by Edmandegradation, which is well known to those of skill in the art. Thepeptide information generated from mass spectrometry or Edmandegradation can be used to design probes or primers that are used toclone the protein of interest.

An alternative method of cloning the protein of interest is by “panning”using EphA2 for cells expressing the antibody or protein of interest.The “panning” procedure is conducted by obtaining a cDNA library fromtissues or cells that express the antibody or protein of interest,over-expressing the cDNAs in a second cell type, and screening thetransfected cells of the second cell type for a specific binding toEphA2. Detailed descriptions of the methods used in cloning mammaliangenes coding for cell surface proteins by “panning” can be found in theart. See, for example, Aruffo, A. and Seed, B. Proc. Natl. Acad. Sci.USA, 84, 8573-8577 (1987) and Stephan, J. et al., Endocrinology 140:5841-5854 (1999).

cDNAs can be obtained by reverse transcribing the mRNAs from aparticular cell type according to standard methods in the art.Specifically, mRNA can be isolated using various lytic enzymes orchemical solutions according to the procedures set forth in Sambrook, etal. supra or extracted by commercially available nucleic-acid-bindingresins following the accompanying instructions provided by manufacturers(e.g., Qiagen, Invitrogen, Promega). The synthesized cDNAs are thenintroduced into an expression vector to produce the antibody or proteinof interest in cells of a second type. It is implied that an expressionvector must be replicable in the host cells either as episomes or as anintegral part of the chromosomal DNA. Suitable expression vectorsinclude but are not limited to plasmids, viral vectors, includingadenoviruses, adeno-associated viruses, retroviruses, and cosmids.

The vectors containing the polynucleotides of interest can be introducedinto the host cell by any of a number of appropriate means, includingelectroporation, transfection employing calcium chloride, rubidiumchloride, calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; and infection (e.g., where thevector is an infectious agent such as vaccinia virus). The choice ofintroducing vectors or polynucleotides will often depend on features ofthe host cell.

Any host cells capable of over-expressing heterologous DNAs can be usedfor the purpose of isolating the genes encoding the antibody,polypeptide or protein of interest. Non-limiting examples of mammalianhost cells include but not limited to COS, HeLa, and CHO cells.Preferably, the host cells express the cDNAs at a level of about 5 foldhigher, more preferably 10 fold higher, even more preferably 20 foldhigher than that of the corresponding endogenous antibody or protein ofinterest, if present, in the host cells. Screening the host cells for aspecific binding to EphA2 is effected by an immunoassay or FACS. A cellover-expressing the antibody or protein of interest can be identified.

The invention includes polypeptides comprising an amino acid sequence ofthe antibodies of this invention, such as LUCA19, SPL1, SG5 or LUCA40.The polypeptides of this invention can be made by procedures known inthe art. The polypeptides can be produced by proteolytic or otherdegradation of the antibodies, by recombinant methods (i.e., single orfusion polypeptides) as described above or by chemical synthesis.Polypeptides of the antibodies, especially shorter polypeptides up toabout 50 amino acids, are conveniently made by chemical synthesis.Methods of chemical synthesis are known in the art and are commerciallyavailable. For example, a LUCA19, SPL1, SG5 or LUCA40 polypeptide couldbe produced by an automated polypeptide synthesizer employing the solidphase method.

The invention also encompasses single chain variable region fragments(“scFv”) of antibodies of this invention, such as LUCA19, SPL1, SG5 orLUCA40. Single chain variable region fragments are made by linking lightand/or heavy chain variable regions by using a short linking peptide.Bird et al. (1988) Science 242: 423-426. For example a linking peptidecan bridge approximately 3.5 nm between the carboxy terminus of onevariable region and the amino terminus of the other variable region.Linkers can in turn be modified for additional functions, such asattachment of drugs or attachment to solid supports. The single chainvariants can be produced either recombinantly or synthetically. Forsynthetic production of scFv, an automated synthesizer can be used. Forrecombinant production of scFv, a suitable plasmid containingpolynucleotide that encodes the scFv can be introduced into a suitablehost cell, either eukaryotic, such as yeast, plant, insect or mammaliancells, or prokaryotic, such as E. coli. Polynucleotides encoding thescFv of interest can be made by routine manipulations such as ligationof polynucleotides. The resultant scFv can be isolated using standardprotein purification techniques known in the art.

The invention includes modifications to antibodies, such as anti-EphA2antibody, including functionally equivalent antibodies and polypeptidesof LUCA19, SPL1, SG5 or LUCA40 that do not significantly affect theirproperties and variants which have enhanced or decreased activity.Modification of polypeptides is routine practice in the art and need notbe described in detail herein. Examples of modified polypeptides includepolypeptides with conservative substitutions of amino acid residues, oneor more deletions or additions of amino acids which do not significantlydeleteriously change the functional activity, or use of chemicalanalogs. Amino acid residues which can be conservatively substituted forone another include but are not limited to: glycine/alanine;valine/isoleucine/leucine; asparagine/glutamine; aspartic acid/glutamicacid; serine/threonine; lysine/arginine; and phenylalanine/tryosine.These polypeptides also include glycosylated and nonglycosylatedpolypeptides, as well as polypeptides with other post-translationalmodifications, such as, for example, glycosylation with differentsugars, acetylation, and phosphorylation. Preferably, the amino acidsubstitutions would be conservative, i.e., the substituted amino acidwould possess similar chemical properties as that of the original aminoacid. Such conservative substitutions are known in the art, and exampleshave been provided above. Amino acid modifications can range fromchanging or modifying one or more amino acids to complete redesign of aregion, such as the variable region. Changes in the variable region canalter binding affinity and/or specificity. Other methods of modificationinclude using coupling techniques known in the art, including, but notlimited to, enzymatic means, oxidative substitution and chelation.Modifications can be used, for example, for attachment of labels forimmunoassay, such as the attachment of radioactive moieties forradioimmunoassay. Modified LUCA19, SPL1, SG5 or LUCA40 polypeptides aremade using established procedures in the art and can be screened usingstandard assays known in the art, some of which are described below andin the Examples.

The invention also encompasses fusion proteins comprising one or morefragments or regions from the antibodies of this invention, such asLUCA19, SPL1, SG5 or LUCA40. In one embodiment, a fusion polypeptide isprovided that comprises at least 10 contiguous amino acids of variablelight chain region and at least 10 amino acids of variable heavy chainregion. In another embodiment, the fusion polypeptide contains aheterologous immunoglobulin constant region. In another embodiment, thefusion polypeptide contains a light chain variable region and a heavychain variable region of LUCA19, SPL1, SG5 or LUCA40. For purposes ofthis invention, a LUCA19, SPL1, SG5 or LUCA40 fusion protein containsone or more LUCA19, SPL1, SG5 or LUCA40 polypeptides and another aminoacid sequence to which it is not attached in the native molecule, forexample, a heterologous sequence or a homologous sequence from anotherregion. A LUCA19, SPL1, SG5 or LUCA40 polypeptide can be created bymethods known in the art, for example, synthetically or recombinantly.

In another embodiment, LUCA19, SPL1, SG5 or LUCA40 chimeras are providedin which the heavy and/or light chains are fusion proteins. In someembodiments, the constant domain of the chains is from one particularspecies and/or class, and the variable domains are from a differentspecies and/or class. For instance, a chimeric antibody (in someembodiments) is one in which the constant regions are derived from humanorigin, and the variable regions are homologous or derived from LUCA19,SPL1, SG5 or LUCA40 (i.e., murine). Also embodied within the inventionis an antibody with a humanized variable region, in which (in someembodiments) the CDR regions comprise LUCA19, SPL1, SG5 or LUCA40 aminoacid sequences, while the framework regions are derived from humansequences. Other forms of humanized antibodies are known in the art anddescribed herein. Also embodied are functional fragments of chimeras. Anexample is a humanized Fab fragment, which contains a human hingeregion, a human first constant region, a human kappa light or heavychain constant region, and the variable region of light and/or heavychain from LUCA19, SPL1, SG5 or LUCA40. The humanized LUCA19, SPL1, SG5or LUCA40 Fab fragments can in turn be made to form Fab dimers.Typically, the LUCA19, SPL1, SG5 or LUCA40 fusion proteins and LUCA19,SPL1, SG5 or LUCA40 chimeras of this invention are made by preparing anexpressing a polynucleotide encoding them using recombinant methodsdescribed herein, although they may also be prepared by other meansknown in the art, including, for example, chemical synthesis. See, forexample, U.S. Pat. Nos. 5,807,715; 4,816,567; and 6,331,415.

The invention also encompasses humanized antibodies. The polynucleotidesequence of an antibody, such as LUCA19, SPL1, SG5 or LUCA40 or otherequivalent antibodies may be used for genetic manipulation to generate a“humanized” antibody, or to improve the affinity, or othercharacteristics of the antibody. The general principle in humanizing anantibody involves retaining the basic sequence of the antigen-bindingportion of the antibody, while swapping the non-human remainder of theantibody with human antibody sequences. There are four general steps tohumanize a monoclonal antibody. These are: (1) determining thenucleotide and predicted amino acid sequence of the starting antibodylight and heavy variable domains (2) designing the humanized antibody,i.e., deciding which antibody framework region to use during thehumanizing process (3) the actual humanizing methodologies/techniquesand (4) the transfection and expression of the humanized antibody. Forexample, the constant region may be engineered to more resemble humanconstant regions to avoid immune response if the antibody is used inclinical trials and treatments in humans. See, for example, U.S. Pat.Nos. 5,997,867 and 5,866,692.

A number of “humanized” antibody molecules comprising an antigen-bindingsite derived from a non-human immunoglobulin have been described,including chimeric antibodies having rodent V regions and theirassociated complementarity determining regions (CDRs) fused to humanconstant domains. See, for example, Winter et al. Nature 349:293-299(1991); Lobuglio et al. Proc. Nat. Acad. Sci. USA 86:4220-4224 (1989);Shaw et al. J Immunol. 138:4534-4538 (1987); and Brown et al. CancerRes. 47:3577-3583 (1987). Other references describe rodent CDRs graftedinto a human supporting framework region (FR) prior to fusion with anappropriate human antibody constant domain. See, for example, Riechmannet al. Nature 332:323-327 (1988); Verhoeyen et al. Science 239:1534-1536(1988); and Jones et al. Nature 321:522-525 (1986). Another referencedescribes rodent CDRs supported by recombinantly veneered rodentframework regions. See, for example, European Patent Publication No.519,596. These “humanized” molecules are designed to minimize unwantedimmunological response toward rodent antihuman antibody molecules thatlimits the duration and effectiveness of therapeutic applications ofthose moieties in human recipients. Other methods of humanizingantibodies that may also be utilized are disclosed by Daugherty et al.,Nucl. Acids Res., 19:2471-2476 (1991) and in U.S. Pat. Nos. 6,180,377;6,054,297; 5,997,867; 5,866,692; 6,210,671; 6,350,861; and PCT WO01/27160.

In yet another alternative, fully human antibodies may be obtained byusing commercially available mice that have been engineered to expressspecific human immunoglobulin proteins. Transgenic animals that aredesigned to produce a more desirable (e.g., fully human antibodies) ormore robust immune response may also be used for generation of humanizedor human antibodies. Examples of such technology are Xenomouse™ fromAbgenix, Inc. (Fremont, Calif.) and HuMAb-Mouse® and TC Mouse™ fromMedarex, Inc. (Princeton, N.J.).

This invention also provides compositions comprising LUCA19, SPL1, SG5or LUCA40 or LUCA19, SPL1, SG5 or LUCA40 equivalent antibodies orpolypeptides conjugated (for example, linked) to a therapeutic agent,such as a radioactive molecule, a toxin (e.g., calicheamicin), or achemotherapeutic molecule, or to liposomes or other vesicles containingchemotherapeutic compounds. The compositions, when administered to anindividual, can target these agents to a cancer cell expressing EphA2recognized by the antibody or polypeptide(s) and thus can, for example,eliminate cancerous cells and/or suppress proliferation and/or growth ofcancerous cells. For simplicity, reference will be made generally toLUCA19, SPL1, SG5 or LUCA40 or antibodies with the understanding thatthese methods apply to any of the EphA2 binding embodiments describedherein. With these methods, conjugation generally refers to linkingthese components as described herein. The linking (which is generallyfixing these components in proximate association at least foradministration) can be achieved in any number of ways, as describedbelow.

A radioactive molecule of this invention includes any radioisotope thatis effective in destroying a cancerous cell. Examples include, but notlimited to, cobalt-60 and X-rays. Additionally, naturally occurringradioactive elements such as uranium, radium, and thorium that typicallyrepresent mixtures of radioisotopes, are suitable examples of aradioactive molecule.

A toxin of the invention include, but not limited to, taxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, tenoposide, vincristine, vinblastine, Colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. The antibodies of the invention can be internalizedwithin the carcinoma cells to which they bind and are thereforeparticularly useful for therapeutic applications, for example,delivering into the cells toxins that need to be internalized for theiradverse activity. Examples of such toxins include, but not limited to,saporin, calicheamicin, auristatin, and maytansinoid.

The antibodies or polypeptides of the invention can be conjugated(linked) to a radioactive molecule, a toxin, or other therapeuticagents, or to liposomes or other vesicles containing therapeutic agentscovalently or non-covalently, directly or indirectly. The antibody maybe linked to the radioactive molecule, the toxin, or the therapeuticmolecule at any location along the antibody so long as the antibody isable to bind its target EphA2.

A toxin or a therapeutic agent may be coupled (e.g., covalently bonded)to a suitable monoclonal antibody either directly or indirectly (e.g.,via a linker group, or, alternatively, via a linking molecule withappropriate attachment sites, such as a platform molecule as describedin U.S. Pat. No. 5,552,391). The toxin and therapeutic agent of thepresent invention can be coupled directly to the particular targetingproteins using methods known in the art. For example, a direct reactionbetween an agent and an antibody is possible when each possesses asubstituent capable of reacting with the other. For example, anucleophilic group, such as an amino or sulfhydryl group, on one may becapable of reacting with a carbonyl-containing group, such as ananhydride or an acid halide, or with an alkyl group containing a goodleaving group (e.g., a halide) on the other.

The antibodies or polypeptides can also be linked to a therapeutic agentvia a microcarrier. Microcarrier refers to a biodegradable or anon-biodegradable particle which is insoluble in water and which has asize of less than about 150, 120 or 100 mm in size, more commonly lessthan about 50-60 μm, preferably less than about 10, 5, 2.5, 2 or 1.5 μm.Microcarriers include “nanocarriers”, which are microcarriers have asize of less than about 1 μm, preferably less than about 500 nm. Suchparticles are known in the art. Solid phase microcarriers may beparticles formed from biocompatible naturally occurring polymers,synthetic polymers or synthetic copolymers, which may include or excludemicrocarriers formed from agarose or cross-linked agarose, as well asother biodegradable materials known in the art. Biodegradable solidphase microcarriers may be formed from polymers which are degradable(e.g., poly(lactic acid), poly(glycolic acid) and copolymers thereof) orerodible (e.g., poly(ortho esters such as3,9-diethylidene-2,4,8,10-tetraoxaspiro[5.5]undecane (DETOSU) orpoly(anhydrides), such as poly(anhydrides) of sebacic acid) undermammalian physiological conditions. Microcarriers may also be liquidphase (e.g., oil or lipid based), such liposomes, iscoms(immune-stimulating complexes, which are stable complexes ofcholesterol, and phospholipid, adjuvant-active saponin) without antigen,or droplets or micelles found in oil-in-water or water-in-oil emulsions,provided the liquid phase microcarriers are biodegradable. Biodegradableliquid phase microcarriers typically incorporate a biodegradable oil, anumber of which are known in the art, including squalene and vegetableoils. Microcarriers are typically spherical in shape, but microcarriersthat deviate from spherical shape are also acceptable (e.g., ellipsoid,rod-shaped, etc.). Due to their insoluble nature (with respect towater), microcarriers are filterable from water and water-based(aqueous) solutions.

The antibody or polypeptide conjugates of the present invention mayinclude a bifunctional linker that contains both a group capable ofcoupling to a toxic agent or therapeutic agent and a group capable ofcoupling to the antibody. A linker can function as a spacer to distancean antibody from an agent in order to avoid interference with bindingcapabilities. A linker can be cleavable or non-cleavable. A linker canalso serve to increase the chemical reactivity of a substituent on anagent or an antibody, and thus increase the coupling efficiency. Anincrease in chemical reactivity may also facilitate the use of agents,or functional groups on agents, which otherwise would not be possible.The bifunctional linker can be coupled to the antibody by means that areknown in the art. For example, a linker containing an active estermoiety, such as an N-hydroxysuccinimide ester, can be used for couplingto lysine residues in the antibody via an amide linkage. In anotherexample, a linker containing a nucleophilic amine or hydrazine residuecan be coupled to aldehyde groups produced by glycolytic oxidation ofantibody carbohydrate residues. In addition to these direct methods ofcoupling, the linker can be indirectly coupled to the antibody by meansof an intermediate carrier such as an aminodextran. In these embodimentsthe modified linkage is via either lysine, carbohydrate, or anintermediate carrier. In one embodiment, the linker is coupledsite-selectively to free thiol residues in the protein. Moieties thatare suitable for selective coupling to thiol groups on proteins are wellknown in the art. Examples include disulfide compounds, α-halocarbonyland α-halocarboxyl compounds, and maleimides. When a nucleophilic aminefunction is present in the same molecule as an α-halo carbonyl orcarboxyl group the potential exists for cyclization to occur viaintramolecular alkylation of the amine. Methods to prevent this problemare well known to one of ordinary skill in the art, for example bypreparation of molecules in which the amine and α-halo functions areseparated by inflexible groups, such as aryl groups or trans-alkenes,that make the undesired cyclization stereochemically disfavored. See,for example, U.S. Pat. No. 6,441,163 for preparation of conjugates ofmaytansinoids and antibody via a disulfide moiety.

One of the cleavable linkers that can be used for the preparation ofantibody-drug conjugates is an acid-labile linker based on cis-aconiticacid that takes advantage of the acidic environment of differentintracellular compartments such as the endosomes encountered duringreceptor mediated endocytosis and the lysosomes. See, for example, Shenet al., Biochem. Biophys. Res. Commun. 102:1048-1054 (1981) for thepreparation of conjugates of daunorubicin with macromolecular carriers;Yang et al., J. Natl. Canc. Inst. 80:1154-1159 (1988) for thepreparation of conjugates of daunorubicin to an anti-melanoma antibody;Dillman et al., Cancer Res. 48:6097-6102 (1988) for using an acid-labilelinker in a similar fashion to prepare conjugates of daunorubicin withan anti-T cell antibody; Trouet et al., Proc. Natl. Acad. Sci.79:626-629 (1982) for linking daunorubicin to an antibody via a peptidespacer arm.

An antibody (or polypeptide) of this invention may be conjugated(linked) to a radioactive molecule by any method known to the art. For adiscussion of methods for radiolabeling antibody see “Cancer Therapywith Monoclonal AntibodiesT”, D. M. Goldenberg ed. (CRC Press, BocaRaton, 1995).

An antibody (or polypeptide) of this invention may be linked to alabeling agent (alternatively termed “label”) such as a fluorescentmolecule, a radioactive molecule or any others labels known in the art.Labels are known in the art that generally provide (either directly orindirectly) a signal.

The ability of the antibodies, polypeptides and proteins of thisinvention, such as ability to inhibit growth of cancerous cellsexpressing EphA2, ability to delay development of metastasis in anindividual with cancer expressing EphA2, ability to deliver atherapeutic agent, such as a toxin, or a radioactive compound, tocancerous cells expressing EphA2, including ability to deliver atherapeutic agent into cancerous cells expressing EphA2, may be testedusing methods known in the art, some of which are described in theExamples.

The invention also provides compositions (including pharmaceuticalcompositions) comprising anti-EphA2 antibody or LUCA19, SPL1, SG5 orLUCA40 equivalent antibodies (which, as this disclosure makes clear,include all of the antibodies described herein) or polypeptides and atherapeutic agent.

Methods for Screening Monoclonal Antibodies

Several methods may be used to further screen monoclonal antibodies thatbind to EphA2 on cancerous cells. One method that may be employed isimmunohistochemistry (IHC). Standard immunohistochemical techniques areknown to those of average skill in the art. See, for example, AnimalCell Culture Methods (J. P. Mather and D. Barnes, eds., Academic Press,Vol. 57, Ch. 18 and 19, pp. 314-350, 1998). Biological samples (e.g.,tissues) may be obtained from biopsies, autopsies, or necropsies. Toascertain if EphA2 is present only on cancerous cells, LUCA19, SPL1, SG5or LUCA40 may be used to detect the presence of EphA2 on tissues fromindividuals with cancer while other non-cancerous tissues from theindividual suffering from cancer or tissues from individuals withoutcancer are used as a control. The tissue can be embedded in a solid orsemi-solid substance that prevents damage during freezing (e.g., agarosegel or OCT) and then sectioned for staining. Cancers from differentorgans and at different grades can be used to screen monoclonalantibodies. Examples of tissues that may be used for screening purposesinclude but are not limited to ovary, breast, lung, prostate, colon,kidney, skin, thyroid, brain, heart, liver, stomach, nerve, bloodvessels, bone, upper digestive tract, and pancreas. Examples ofdifferent cancer types that may be used for screening purposes includebut are not limited to carcinomas, adenocarcinomas, sarcomas,adenosarcomas, lymphomas, and leukemias.

In yet another alternative, cancerous cells lines such as SK-Ov-3 (ATCC#HTB 77), LnCap (ATCC #CRL-1740), COLO 205 (ATCC #CCL 222), A549 (ATCC#CCL 185), PANC-1 (ATCC #CRL 1469), SK-BR-3 (ATCC #HTB 30), SK-MES-1(ATCC #HTB 58), HT-29 (HTB-38), SW 480 (ATCC #CCL 228), AsPC-1 (ATCC#CRL 1682), Capan-1 (ATCC #HTB 79), CFPAC-1 (ATCC #CRL 1918), HPAF-II(ATCC #CRL-1997), HS-700T (ATCC #HTB 147), Du-145 (ATCC #HTB-81), CaLu-1(ATCC #HTB-54), 786-0 (ATCC #CRL-1932). CaKi-2 (ATCC #HTB-47), A498(ATCC #HTB-44), BT474 (ATCC #HTB-20), and PC-3 (ATCC #CRL 1435) andnormal cells from their respective tissues may be used to screen formonoclonal antibodies which are specific for cancerous tissue. Primary,or low passage, cell cultures derived from normal tissues from differentorgans, including but not limited to, ovary, breast, lung, prostate,colon, kidney, skin, thyroid, aortic smooth muscle, and endothelialcells can be used as negative controls. The cancerous or non-cancerouscells can be grown on glass slides or coverslips, or on plasticsurfaces, or prepared in a CellArray™, as described in WO 01/43869, andscreened for the binding of antibody using IHC as described above fortissues. Alternatively, cells may be removed from the growth surfaceusing non-proteolytic means and spun into a pellet which is thenembedded and treated as tissues for IHC analysis as described above. Inanother alternative, single cells may be screened by incubating with theprimary antibody, a secondary “reporter” antibody linked to afluorescent molecule and then analyzed using a fluorescent activatedcell-sorting (FACS) machine.

Several different detection systems may be utilized to detect binding ofantibodies to tissue section. Typically, immunohistochemistry involvesthe binding of a primary antibody to the tissue and then a secondaryantibody reactive against the species from the primary antibody wasgenerated and conjugated to a detectable marker (e.g., horseradishperoxidase, HRP, or diaminobenzedine, DAB). One alternative method thatmay be used is polyclonal mirror image complementary antibodies orpolyMICA. PolyMICA (polyclonal Mirror Image Complementary Antibodies)technique, described by D. C. Mangham and P. G. Isaacson (Histopathology(1999) 35(2):129-33), can be used to test binding of primary antibodies(e.g., LUCA19, SPL1, SG5 or LUCA40) to normal and cancerous tissue.Several kinds of polyMICA™ Detection kits are commercially availablefrom The Binding Site Limited (P.O. Box 4073 Birmingham B29 6ATEngland). Product No. HK004.D is a polyMICA™ Detection kit which usesDAB chromagen. Product No. HK004.A is a polyMICA™ Detection kit whichuses AEC chromagen. Alternatively, the primary antibody may be directlylabeled with the detectable marker.

The first step in IHC screening to select for an appropriate antibody isthe binding of primary antibodies raised in mice (e.g., LUCA19, SPL1,SG5 or LUCA40) to one or more immunogens (e.g., cells or tissuesamples). In one embodiment, the tissue sample is sections of frozentissue from different organs. The cells or tissue samples can be eithercancerous or non-cancerous.

Frozen tissues can be prepared, sectioned, with or without fixation, andIHC performed by any of a number of methods known to one familiar withthe art. See, for example, Stephan et al. Dev. Biol. 212: 264-277(1999), and Stephan et al. Endocrinology 140: 5841-54 (1999).

Monoclonal antibodies that are cross-reactive with human cells and thatbind to cancerous cells or tissues, but not to normal cells or tissuesto the same degree, are selected. Monoclonal antibodies that bind toantigens expressed on one or more cancer types but not to normal cellsare also selected. LUCA19, SPL1, SG5 or LUCA40 is an example of anantibody that binds to the antigen EphA2 present on a number ofdifferent cancers, but has limited binding to normal tissues. Epitopemapping may be used to further characterize the antibody. Commerciallyavailable services (e.g., Pepscan Systems, P.O. Box 2098, 8203 ABLelystad, The Netherlands) may be used to determine the epitope(s) onthe antigen EphA2 to which an antibody, such as LUCA19, SPL1, SG5 orLUCA40, binds.

Methods of Diagnosing Cancer Using LUCA19, SPL1, SG5 or LUCA40,Equivalent Antibodies or Polypeptides that Bind to EphA2

Monoclonal anti-EphA2 antibody and equivalent antibodies or polypeptidesderivatives of LUCA19, SPL1, SG5 or LUCA40 which bind EphA2 made by themethods disclosed herein may be used to identify or detect the presenceor absence of cancerous cells in a variety of tissues, including but notlimited to, ovary, breast, lung, prostate, colon, kidney, skin, thyroid,brain, heart, liver, stomach, nerve, blood vessels, bone, upperdigestive tract, and pancreas for purposes of diagnosis. For simplicity,reference will be made generally to LUCA19, SPL1, SG5 or LUCA40 orantibodies with the understanding that these methods apply to any of theEphA2 binding embodiments described herein. Detection generally involvescontacting cells with an antibody or a polypeptide described herein thatbinds to EphA2 and the formation of a complex between EphA2 and anantibody (e.g., LUCA19, SPL1, SG5 or LUCA40, a humanized antibody ofLUCA19, SPL1, SG5 or LUCA40, a human antibody or any other EphA2 bindingmoiety) that binds specifically to EphA2. The formation of such acomplex can be in vitro or in vivo. Without being bound by theory,monoclonal anti-EphA2 antibody can bind to EphA2 through theextracellular domain of EphA2. As used herein, detection may includequalitative and/or quantitative detection and may include comparing thelevel measured to a normal cell for an increased level of expression ofEphA2 in cancerous cells.

One method of using the antibodies for diagnosis is in vivo tumorimaging by linking the antibody to a labeling moiety (e.g., afluorescent agent, a radioactive or radioopaque agent), administeringthe antibody to the patient and using an x-ray or other imaging machineto visualize the localization of the labeled antibody at the surface ofcancer cells expressing the antigen. Labeling moieties are known in theart.

In other methods, the cancerous cells are removed and the tissueprepared for immunohistochemistry by methods well known in the art(e.g., embedding in a freezing compound, freezing and sectioning, withor without fixation; fixation and paraffin embedding with or withoutvarious methods of antigen retrieval and counterstaining). Themonoclonal antibodies may also be used to identify neoplasms atdifferent stages of development. The antibodies may also be used todetermine which patients' tumors express the antigen on their surface ata pre-determined level and are thus candidates for immunotherapy usingantibodies directed against said antigen.

Antibodies (or polypeptides) recognizing the antigen may also be used tocreate diagnostic immunoassays for detecting antigen released orsecreted from living or dying cancer cells in bodily fluids, includingbut not limited to, blood, saliva, urine, pulmonary fluid, or ascitesfluid. As discussed in further detail in the Examples, LUCA19, SPL1, SG5or LUCA40 can bind to various forms cancer in different stages fromtissues including but not limited to melanomas, breast, lung, prostate,and colon. Methods of using LUCA19, SPL1, SG5 or LUCA40 for diagnosticpurposes is useful both before and after any form of anti-cancertreatment, e.g., chemotherapy or radiation therapy, to determine whichtumors are most likely to respond to a given treatment, patientprognosis, tumor subtype or origin of metastatic disease, andprogression of the disease or response to treatment.

Methods of Using LUCA19, SPL1, SG5 or LUCA40, or Equivalent Antibodiesor Polypeptides for Therapeutic Purposes

Monoclonal anti-EphA2 antibody and equivalent antibodies (as well asother polypeptides embodiments of the invention) made by the methodsdisclosed herein may be used for therapeutic purposes in individualswith cancer, including but not limited to melanomas, breast, lung,colon, or prostate cancer. These therapeutic methods also apply to thelinked embodiments described above. For simplicity, reference will bemade generally to LUCA19, SPL1, SG5 or LUCA40 or antibodies with theunderstanding that these methods apply to any of the EphA2 bindingembodiments described herein, including but not limited to humanizedantibodies and human antibodies described herein including linkedembodiments. Therapy with LUCA19, SPL1, SG5 or LUCA40 can involveformation of complexes of such antibody and EphA2 both in vitro and/orin vivo as described above. In one embodiment, monoclonal anti-EphA2antibody can bind to and reduce the growth and/or proliferation ofcancerous cells. In another embodiment, monoclonal anti-EphA2 antibodycan bind to and induce apoptotic cell death in the cancer cell. Inanother embodiment, monoclonal anti-EphA2 antibody can bind to cancerouscells and delay the development of metastasis. In another embodiment,monoclonal anti-EphA2 antibody can bind to cancerous cells and deliver atherapeutic agent (such as a toxin, or a radioactive compound) linked toLUCA19, SPL1, SG5 or LUCA40 to cancerous cells. For some embodiments,therapeutic agent (such as a toxin) is introduced into a cell (i.e., isinternalized). Particularly suitable agents for these methods includeagents that are active inside the cell. Examples of such agents includebut not limited to saporin, calicheamicin, auristatin, and maytansinoid.Generally, in these embodiments an effective amount (an amountsufficient to deliver a therapeutic agent to and into target cancerouscells) is administered to an individual. In yet another embodiment, anindividual with cancer is given palliative treatment with LUCA19, SPL1,SG5 or LUCA40. Palliative treatment of a cancer patient involvestreating or lessening the adverse symptoms of the disease, or iatrogenicsymptoms resulting from other treatments given for the disease withoutdirectly affecting the cancer progression. This includes treatments foreasing of pain, nutritional support, sexual problems, psychologicaldistress, depression, fatigue, psychiatric disorders, nausea, vomiting,etc.

This invention also provides methods of inhibiting growth and/orproliferation of cancer cells using LUCA19, SPL1, SG5 or LUCA40 antibodyor an antibody which preferentially binds to the same epitope as theepitope to which any of LUCA19, SPL1, SG5 or LUCA40 preferentiallybinds.

In yet another embodiment, LUCA19, SPL1, SG5 or LUCA40 or any of theEphA2 embodiments described herein can bind to EphA2 expressingcancerous cells and induces an active immune response against thecancerous cells expressing EphA2. In some cases, the active immuneresponse can cause the death of the cancerous cells (e.g., LUCA19, SPL1,SG5 or LUCA40 binding to cancer cells inducing apoptotic or oncotic celldeath), or inhibit the growth (e.g., block cells cycle progression) ofthe cancerous cells. In other cases, LUCA19, SPL1, SG5 or LUCA40 or anyof the EphA2 antibodies described herein can bind to cancerous cells andantibody-dependent cellular cytotoxicity (ADCC) can eliminate cancerouscells to which LUCA19, SPL1, SG5 or LUCA40 binds. Accordingly, theinvention provides methods of stimulating an immune response comprisingadministering any of the compositions described herein.

The anti-EphA2 antibody may be administered with agents that enhance ordirect an individual's own immune response, such as an agent thatstrengthens ADCC. In one embodiment, at least one fucose residue presentin an anit-EphA2 antibody is removed from the oligosaccharides of thatantibody, a modification to enhance ADCC. In similar embodiments, fucoseresidues present in an anti-EphA2 antibody are modified to alter theircomposition to the extent required to enhance ADCC compared to theoriginal, unmodified antibody.

In some cases, LUCA19, SPL1, SG5 or LUCA40 binding can also activateboth cellular and humoral immune responses and recruit more naturalkiller cells or increased production of cytokines (e.g., IL-2, IFN-g,IL-12, TNF-a, TNF-b, etc.) that further activate an individual's immunesystem to destroy cancerous cells. In yet another embodiment, LUCA19,SPL1, SG5 or LUCA40 can bind to cancerous cells, and macrophages orother phagocytic cell can opsonize the cancerous cells.

In some embodiments, the invention provides methods of conferringpassive immunity comprising administering any of the compositionsdescribed herein.

The invention provides methods of delivering any of the compositions(including conjugates) described herein to an EphA2 expressing cell,such as an EphA2 expression cancer cells. These methods entailadministering the compositions (including conjugates) described hereinto an individual. In some embodiments, the methods provide forintroducing, for example, a conjugate into a target cell. In yet anotherembodiment, LUCA19, SPL1, SG5 or LUCA40 can be conjugated to atherapeutic agent (such as a radioactive molecule, a toxin, e.g.,saporin, calicheamicin, auristatin, or maytansinoid, or otherchemotherapeutic molecule) or to liposomes or other vesicles containingchemotherapeutic compounds and administered to an individual to targetthese compounds to the cancer cell containing the antigen recognized bythe antibody and thus eliminate cancerous cells. In yet anotherembodiment, the antibody can be employed as adjuvant therapy at the timeof the surgical removal of a cancer expressing the antigen in order todelay the development of metastasis. The antibody can also beadministered before surgery (neoadjuvant therapy) in a patient with atumor expressing the antigen in order to decrease the size of the tumorand thus enable or simplify surgery, spare tissue during surgery, and/ordecrease the resulting disfigurement.

Various formulations of LUCA19, SPL1, SG5 or LUCA40 and equivalentantibodies or fragments (e.g., Fab, Fab′, F(ab′)₂, Fv, Fc, etc.), suchas chimeric antibodies, single chain (ScFv), mutants thereof, fusionproteins comprising an antibody portion, humanized antibodies, and anyother modified configuration of LUCA19, SPL1, SG5 or LUCA40 thatcomprises an antigen EphA2 recognition site of the required specificity,may be used for administration. In some embodiments, LUCA19, SPL1, SG5or LUCA40 antibodies or various formulations of LUCA19, SPL1, SG5 orLUCA40 thereof may be administered neat. In other embodiments, LUCA19,SPL1, SG5 or LUCA40 or various formulations of LUCA19, SPL1, SG5 orLUCA40 (including any composition embodiment described herein) thereofand a pharmaceutically acceptable excipient are administered, and may bein various formulations. Pharmaceutically acceptable excipients areknown in the art, and are relatively inert substances that facilitateadministration of a pharmacologically effective substance. For example,an excipient can give form or consistency, or act as a diluent. Suitableexcipients include but are not limited to stabilizing agents, wettingand emulsifying agents, salts for varying osmolarity, encapsulatingagents, buffers, and skin penetration enhancers. Excipients as well asformulations for parenteral and nonparenteral drug delivery are setforth in Remington, The Science and Practice of Pharmacy 20th Ed. MackPublishing (2000).

Generally, these agents are formulated for administration by injection(e.g., intraperitoneally, intravenously, subcutaneously,intramuscularly, etc.), although other forms of administration (e.g.,oral, mucosal, etc) can be also used. Accordingly, LUCA19, SPL1, SG5 orLUCA40 antibody and equivalents thereof are preferably combined withpharmaceutically acceptable vehicles such as saline, Ringer's solution,dextrose solution, and the like. The particular dosage regimen, i.e.,dose, timing and repetition, will depend on the particular individualand that individual's medical history. Generally, any of the followingdoses may be used: a dose of at least about 50 mg/kg body weight; atleast about 10 mg/kg body weight; at least about 3 mg/kg body weight; atleast about 1 mg/kg body weight; at least about 750 μg/kg body weight;at least about 500 μg/kg body weight; at least about 250 ug/kg bodyweight; at least about 100 μg/kg body weight; at least about 50 μg/kgbody weight; at least about 10 ug/kg body weight; at least about 1 μg/kgbody weight, or more, is administered. Empirical considerations, such asthe half-life, generally will contribute to determination of the dosage.Antibodies that are compatible with the human immune system, such ashumanized antibodies or fully human antibodies, may be used to prolonghalf-life of the antibody and to prevent the antibody being attacked bythe host's immune system.

In some individuals, more than one dose may be required. Frequency ofadministration may be determined and adjusted over the course oftherapy, and is based on reducing the number of cancerous cells,maintaining the reduction of cancerous cells, reducing the growth and/orproliferation of cancerous cells, or delaying the development ofmetastasis. The presence of cancerous cells can be identified by anynumber of methods known to one of skill in the art or discussed herein(e.g., detection by immunohistochemistry or flow cytometry of biopsiesor biological samples). In some cases, sustained continuous releaseformulations of LUCA19, SPL1, SG5 or LUCA40 antibodies may beappropriate. Various formulations and devices for achieving sustainedrelease are known in the art.

In one embodiment, dosages for LUCA19, SPL1, SG5 or LUCA40 antibodiesmay be determined empirically in individuals who have been given one ormore administration(s). Individuals are given incremental dosages ofLUCA19, SPL1, SG5 or LUCA40. To assess efficacy of LUCA19, SPL1, SG5 orLUCA40 or other equivalent antibody, markers of the specific cancerdisease state can be monitored. These markers include: directmeasurements of tumor size via palpation or visual observation; indirectmeasurement of tumor size by x-ray or other imaging techniques; animprovement as assessed by direct tumor biopsy and microscopicexamination of the tumor sample; the measurement of an indirect tumormarker (e.g., PSA for prostate cancer), a decrease in pain or paralysis;improved speech, vision, breathing or other disability associated withthe tumor; increased appetite; or an increase in quality of life asmeasured by accepted tests or prolongation of survival. It will beapparent to one of skill in the art that the dosage will vary dependingon the individual, the type of cancer, the stage of cancer, whether thecancer has begun to metastasize to other location in the individual, andthe past and concurrent treatments being used.

Other formulations include suitable delivery forms known in the artincluding, but not limited to, carriers such as liposomes. See, forexample, Mahato et al. (1997) Pharm. Res. 14:853-859. Liposomalpreparations include, but are not limited to, cytofectins, multilamellarvesicles and unilamellar vesicles.

In some embodiments, more than one antibody may be present. Theantibodies can be monoclonal or polyclonal. Such compositions maycontain at least one, at least two, at least three, at least four, atleast five different antibodies that are reactive against carcinomas,adenocarcinomas, sarcomas, or adenosarcomas. LUCA19, SPL1, SG5 or LUCA40antibody can be admixed with one or more antibodies reactive againstcarcinomas, adenocarcinomas, sarcomas, or adenosarcomas in organsincluding but not limited to ovary, breast, lung, prostate, colon,kidney, skin, thyroid, bone, upper digestive tract, and pancreas. Amixture of antibodies, as they are often denoted in the art, may beparticularly useful in treating a broader range of population ofindividuals.

Assessment of disease is performed using standard methods in the arts,such as imaging methods and monitoring appropriate marker(s).

Kits Comprising Antibodies and Polypeptides of the Invention that Bindto EphA2

The invention also provides kits comprising antibodies or any of thecompositions described herein that bind to EphA2 for use in diagnosisand/or therapy. Accordingly, the kits comprise an antibody that can bindto EphA2 preferentially and/or form a complex with EphA2 (useful, forexample, for detecting breast, colon, lung, or prostate cancerouscells). In some embodiments, the kits comprise anti-EphA2 antibody or anantibody that preferentially binds to the same epitope as LUCA19, SPL1,SG5 or LUCA40 preferentially binds. In some embodiments, the kitscomprise anti-EphA2 antibody or an antibody that preferentially binds tothe same epitope as LUCA19, SPL1, SG5 or LUCA40 preferentially bindslinked to a therapeutic agent or a labeling agent. These kits mayfurther include instruction and/or reagents for linking the antibody orany antibody or polypeptide embodiments described herein to thetherapeutic agent(s) or the labeling agent(s). In some aspects, thebinding of an antibody (e.g., monoclonal, polyclonal, human, humanized,etc.) to EphA2 is used for diagnosing cancer in an individual, forexample, kits for detecting presence or absence of cancerous cells, andkits for detecting presence or absence of breast, colon, lung, orprostate cancerous cells. In other aspects, the kits may be used, forexample, to treat an individual with cancer or a family history ofcancer. Kits for treating individual with cancer include but not limitedto kits for inhibiting growth and/or proliferation of cancer cells, fordelivering a therapeutic agent to cancerous cells, for delivering atherapeutic agent into cancerous cells. The kits of this invention arein suitable packaging, and may optionally provide additional componentssuch as, buffers and instructions for determining binding to EphA2, suchas capture reagents, developing reagents, labels, reacting surfaces,means for detection, control samples, and interpretive information. Theinstructions may be for any measurement of antigen binding, including,but not limited to, those assays described herein. In other embodiments,the instructions may be for any of the methods described herein,including: instructions for inhibiting grow and/or proliferation ofcancerous cells such as breast, lung, colon, or prostate, for deliveringa therapeutic agent to cancerous cells, for delivering a therapeuticagent into cancerous cells. In some embodiments, reagents describedabove are supplied such that multiple measurements may be made, such asallowing for measurements in the same individual over time or multipleindividuals. Any appropriate means for detecting binding of theantibodies may be employed (and provided in the kits) such as a labeledanti-human antibody, wherein the label may be an enzyme, fluorophore,chemiluminescent material radioisotope or coenzyme. Generally, the labelused will be an enzyme.

The following examples are provided to illustrate, but not to limit, theinvention.

EXAMPLES Example 1 Preparation of Cancer Cell Lines as an Immunogen

Whole cells isolated from tissue or from cell culture were used as animmunogen for producing monoclonal antibodies that are specific forsurface antigens representative of a particular cell type. Such methods,suitable for the practice of this invention, are described in U.S. Pat.No. 6,541,225. Generally, to produce monoclonal antibodies directed tocell-surface antigens of a specific cell type, it is desirable toimmunize non-transformed B-cells with viable and intact cells of thattype, preferably with those cells whose surfaces that are free of serum.Cell lines that are suitable for the generation of monoclonal antibodiesagainst the antigen EphA2, such as but not limited to LUCA19, LUCA 40,or SPL1, include: BT-474 (ATCC #HTB-20), MDA-MB-175VII (ATCC #HB-25),MDA-MB-361 (ATCC #HB-27), SKBR3 (ATCC #HTB-30), SKMES-1 (ATCC #HTB-58),ES-2 (ATCC #CRL-1978), SKOV3 (ATCC #HTB-77), HPAFII (ATCC #CRL-1997),Hs700T (ATCC #HTB-147), Colo205 (ATCC #CCL-222), HT-29 (ATCC #HTB-38),SW480 (ATCC #CCL-228), SW948 (ATCC #CCL-237), A498 (ATCC #HTB-44) andCaki-2 (ATCC #HTB-47).

The cells were grown in the appropriate nutrient media supplemented withgrowth factors, but free of serum. Immunization with cells that havebeen propagated in a serum-supplemented medium can have extremedisadvantages. Serum contains a complex mixture of small and largebiomolecules with undefined activities. These biomolecules can adhere tothe surfaces of cells and thereby leading to the generation ofantibodies cross-reacting with molecules not representative of thespecific cell type. Additionally, binding of serum biomolecules to thecell surface may lead to the masking of desired cell surface antigentargets. A number of serum-free media preparations are commerciallyknown and publicly available, such as for example, F12/DME (1:1)nutrient media with the following supplements: insulin (10 μg/ml finalconcentration), epidermal growth factor (EGF) (5 ng/ml finalconcentration), selenious acid (2.5×10⁻⁸ M final concentration), andporcine pituitary extract (PPE) (5 μl/ml final concentration).

To harvest the cells, the cell monolayers were rinsed once with calcium-and magnesium-free Hanks saline solution, incubated in 10 mM EDTA inHanks saline solution at 37 C for 15 minutes. The cells were detachedfrom the culture surface by gentle pipetting. The cell suspension waspelleted by centrifugation at 1000×g for 5 minutes. The supernatant wasremoved and cells were resuspended in serum-free medium withnon-denaturing adjuvant as appropriate.

Example 2 Generation of Monoclonal Antibodies

A non-denaturing adjuvant (Ribi, R730, Corixa, Hamilton Mont.) wasrehydrated to 2 ml in phosphate buffered saline. 100 μl of thisrehydrated adjuvant was then gently mixed with some of the cell pelletfrom Example 1 to be used for immunization. Approximately 10⁶ cells permouse were injected into Balb/c mice via footpad, approximately once ortwice a week. The precise immunization schedule is as follows: Day zero,immunization plus Ribi. Day 3, immunization plus Ribi. Day 7,immunization plus Ribi. Day 24, immunization minus Ribi. Day 29,immunization minus Ribi. Day 32, immunization minus Ribi. Day 36,immunization minus Ribi. Day 44, immunization minus Ribi. Day 51,immunization minus Ribi. Day 69, bleed for titer test. Day 71.immunization plus Ribi. Day 74, immunization plus Ribi. Day 81,immunization plus Ribi. Day 91, pre-fusion boost (no Ribi). Day 104,harvest nodes for fusion.

At Day 69, a drop of blood was drawn from the tail of each immunizedanimal to test the titer of antibodies against the cell line used toimmunize using FACS analysis. When the titer reached at least 1:2000,the mice were sacrificed using CO₂ followed by cervical dislocation.Lymph nodes were harvested for hybridoma preparation.

Lymphocytes from mice were fused with the mouse myeloma line X63-Ag8.653using 35% polyethylene glycol 4000. On day 10 following the fusion, thehybridoma supernatants were screened for the presence of the immunizingcells-specific monoclonal antibodies by fluorescence activated cellsorting (FACS). Conditioned medium from each hybridoma was incubated for30 minutes with an aliquot of human fetal kidney cells. Afterincubation, the cell samples were washed, resuspended in 0.1 ml diluentand incubated with 1 μg/ml of FITC conjugated F(ab′)2 fragment of goatanti-mouse IgG for 30 min at 4° C. The cells were washed, resuspended in0.2 ml FACS diluent and analyzed using a FACScan cell analyzer (BectonDickinson; San Jose, Calif.). Hybridoma clones were selected for furtherexpansion, cloning, and characterization based on their binding to thesurface of the human fetal kidney cells by FACS. A hybridoma making amonoclonal antibody designated mu-SPL1 which binds an antigen designatedAg-SPL1 and an epitope on that antigen designated Ag-SPL1.1 wasselected. A hybridoma making monoclonal antibody designated mu-LUCA19which binds an antigen designated Ag-LUCA19 and an epitope on thatantigen designated Ag-LUCA19.1 was selected. A hybridoma makingmonoclonal antibody designated mu-LUCA40 that binds an antigendesignated Ag-LUCA40 and an epitope on that antigen designatedAg-LUCA40.1. A hybridoma making monoclonal antibody designated mu-SG5that binds an antigen designated Ag-SG5 and an epitope on that antigendesignated Ag-SG5.1. The hybridomas that produce the monoclonalantibodies, mu-SPL-1, mu-LUCA19, mu-LUCA40 and mu-SG5 were expanded inculture for purification of the monoclonal antibodies in a culturemedium that is able to support monoclonal antibody growth and antibodypurification.

Example 3 Purification of Anti-EphA2 Antibodies

Human cancer cells such as but not limited to SKMES-1, 786-O, andColo205 cell lines were detached from tissue culture flasks in thepresence of 10.0 mM EDTA, centrifuged at 1400 rpm for 5 minutes andresuspended in PBS containing 1% BSA and 2 mM EDTA (FACS diluent). Thecells were counted and adjusted to 10⁷ cells/ml. About 0.1 ml of cellswere incubated with 100 μl FACS diluent for 30 minutes at 37° C.Monoclonal antibodies that bind to the human cancer cell lines werepurified from tissue culture supernatant using protein-G affinitychromatography. If needed the tissue culture supernatant may be passedover a bovine IgG column before antibody purification in order to removeexcess bovine IgG. The following materials were used for the antibodypurification process: hybridoma tissue culture supernatant, Immunopure(G) IgG binding buffer (Pierce #21011 Rockford, Ill.), Immunopure IgGElution Buffer (Pierce #21009), concentrated HCl (for adjusting pH),Corning 1 liter PES (polyether sulfone), 0.22 μm filter (Corning#431098, Corning, N.Y.), Amersham Pharmacia AKTA Explorer System(Amersham Biosciences, Piscataway, N.J.), Protein-G Sepharose 4 FastFlow (Amersham Biosciences #17-0618-03), Stripping buffer consisting of3M Potassium thiocyanate/50 mM Tris pH 7.8, and PBS (phosphate bufferedsaline), 3M Tris pH 9.0.

To purify the mouse anti-human SPL1, mouse anti-human LUCA19 and mouseanti-human LUCA40 antibodies referred to herein as mu-SPL1, mu-LUCA19and mu-LUCA40, respectively, the volume of the supernatant was measuredand an equal volume of binding buffer was added to the supernatant. Themixture was allowed to equilibrate to room temperature. The supernatantwas clarified by passage through a 0.22 μM filter. The supernatant wasloaded onto a protein-G Sepharose column using the AKTA Explorer system(Amersham Biosciences) and then washed with 5-10 column volumes ofbinding buffer. The monoclonal antibody was eluted with the elutionbuffer, and fractions were collected. The fractions were neutralizedupon elution with the addition of 3M Tris, pH 9.0 to empty tubes ( 1/60volume of the fractions). The peak fractions containing the monoclonalantibody were pooled. The pooled samples was injected into a pre-wettedslidealyzer cassette (10,000 MW cutoff; Pierce #66810) and dialyzed in1× PBS at 4° C. (with 3 buffer changes of at least 4 hours of dialysisper change). The purified monoclonal antibody was sterile filtered (0.2μm Acrodisc) and stored at 2-8° C.

A sample of purified antibody is taken for determination ofconcentration by UV absorbance (A₂₈₀) and SDS-Polyacrylamide gelelectrophoresis (SDS-PAGE). SDS-PAGE is run under both non-reducing andreducing conditions for analysis of molecular weight, identification ofthe typical banding pattern of monoclonal antibodies and assessment ofpurity.

After purification of the mu-SPL1, mu-LUCA19 and mu-LUCA40 monoclonalantibody from the hybridoma supernatant, it was re-tested for binding torespective immunizing or target cells of interest. The cell samples wereprepared as described above and incubated with the purified antibody atvarious concentrations. After incubation the cells were washed,resuspended in 0.1 ml diluent and incubated with 1 μg of FITC conjugatedF(ab)′2 fragment of goat anti-mouse IgG for 30 minutes at 4° C. Thecells were washed, resuspended in 0.5 ml FACS diluent and analyzed usinga FACScan cell sorter (Becton Dickinson, San Jose, Calif.). A shift tothe right on the FACScan histogram indicated that the purified antibodystill bound to the cells.

In other experiments, the binding of the mu-SPL1, mu-LUCA19 andmu-LUCA40 antibodies to SPL1, LUCA19 and LUCA40, respectively, wastested and confirmed using live cell ELISA. the following method wasused, although other methods commonly known in the field are applicable.Cells (HT-29, SKOV3, SKMES-1, SW480, SKBR-3, and HPAFII) were grown in10% fetal bovine serum (FBS) containing media to confluency on tissueculture treated 96-well plates (Falcon). Cells were washed with PBS andthen incubated with 50 μl of desired antibodies at a desiredconcentration in Hank's Balanced Salt Solution (HBSS) containing 1% BSAand 0.1% sodium azide for 1 hour at room temperature. The cells werethen washed three times with 100 μl per well of HBSS before incubationwith horseradish peroxidase (HRP) secondary antibody (50 μl per welldiluted in HBSS) for 30 minutes at room temperature. The cells werefinally washed three times with HBSS and the color change substrate (TMBsubstrate, KPL) was added to each well at 100 μl per well. The colorchange reaction was stopped with the addition of 100 μl per well of 1Mphosphoric acid. The plates were then read at O.D. 450 nm.

Example 4 Western Blot Analysis of SPL1, LUCA19 and LLICA40 Expressionin Cancer Cell Line SW480

Renal cell carcinoma cell line SW480 (ATCC #CCL-228) were grown toconfluency on 175 cm² culture dishes. The confluent monolayer was washedthree times with Hank's Balanced Salt Solution (HBSS+ containing nosodium bicarbonate or phenol red; buffered with 10 mM HEPES, pH 7.4;Sigma Chemicals) and biotinylated with 200 μg of sulfo-NHS-LC-biotin(Pierce Endogen) for 30 minutes at room temperature. The cells were thenwashed with HBSS+ containing 0.1M Tris, pH 7.4 (Sigma Chemicals) andincubated in HBSS+ containing 0.1M Tris, pH 7.4 for 15 minutes at roomtemperature. The cells were finally washed three times with HBSS+ andlysed by incubation for 5 minutes, on ice, in lysis buffer (HBSS+ with2% Triton X-100, 2 mM PMSF, 0.1% sodium azide, and 1 tablet per 5 mllysis buffer of EDTA free complete mini-protease cocktail (RocheMolecular Biochemicals)).

Cells were scraped in lysis buffer and lysates collected. Lysates werecentrifuged at 14,000-×g for one hour at 4° C. The clarified lysate wasthen pre-cleared for 2 hours at 4° C. with 5 μl of human IgG conjugated(1mg/ml) CNBr 4MB sepharose beads (Amersham Pharmacia). Human IgG beadswere centrifuged and removed, and then the pre-cleared lysate was thenincubated with monoclonal antibody mu-SPL1, mu-LUCA19, mu-SG5 ormu-LUCA40 conjugated to CNBr 4MB Sepharose beads (conjugated at 1 mg/ml)for 2 hours at 4° C. The mu-SPL1, mu-LUCA19, mu-SG5 or mu-LUCA40 beadswere centrifuged and removed after the 2-hour incubation. Both the humanIgG and the mu-SPL1, mu-LUCA19, mu-SG5 or mu-LUCA40 beads wereindividually washed three times with 1 ml of lysis buffer and thenwashed three times with 1 ml HBSS+. The washed beads were eluted by theaddition of 30 μl of SDS-PAGE sample buffer and boiling at 99° C. for 5minutes.

The samples were then resolved on a 4-20% Novex gradient gel(Invitrogen), and transferred onto 0.2 μm nitrocellulose membrane(Invitrogen) and visualized by horse radish peroxidase (HRP) conjugatedstreptavidin (Pierce Endogen) or western blotted with 5 μg/blot ofmu-SPL1, mu-LUCA19, mu-SG5 or mu-LUCA40.

For detection with HRP conjugated streptavidin, the nitrocellulose wasfirst blocked for 1 hour with blocking buffer (5% non-fat dry milk inTris-buffered saline with 0.05% Tween-20 (TBST)). HRP conjugatedstreptavidin was diluted into TBST at 1 μg/ml and exposed to thenitrocellulose for 30 minutes at room temperature. The nitrocellulosewas washed three times in TBST before visualization with ECL+(Amersham).

For western blotting with mu-SPL1, mu-LUCA19, mu-SG5 or mu-LUCA40, thenitrocellulose was similarly blocked for 1 hour in blocking buffer. Thenitrocellulose was then incubated in a heat sealed plastic pouchcontaining 1 ml of 5 μg/ml mu-SPL1, mu-LUCA19, mu-SG5 or mu-LUCA40diluted in blocking buffer. The nitrocellulose was washed 3 times withTBST before incubation with 10 ml of 1 μg/ml HRP conjugated donkeyanti-mouse IgG (heavy and light chain specific, cross adsorbed againstbovine, chicken, goat, guinea pig, Syrian hamsters, horse, human,rabbit, sheep serum proteins; Jackson Immunoresearch Cat. #709-035-149)for 1 hour at room temperature. The nitrocellulose was finally washedthree times with TBST and visualized by ECL+ (Amersham).

Example 5 Immunohistochemistry Methods

Frozen tissue samples from cancer patients were embedded in OCT compoundand quick-frozen in isopentane with dry ice. Cryosections were cut witha Leica 3050 CM microtome at thickness of 8-10 μm and thaw-mounted onSuperFrost Plus slides (VWR #48311-703). The sections were fixed with75% acetone/25% ethanol at 10° C. and allowed to air-dry 2-4 hours atroom temperature. The fixed sections were stored at −80° C. until use.

For immunohistochemistry, the tissue sections were retrieved washed inTris buffered 0.05% Tween (TB-T) and blocked in blocking buffer (TB-T,5% normal goat serum and 100 μg/ml avidin) for 30 minutes at roomtemperature. The slides were then incubated with the mu-LUCA19,mu-LUCA40, mu-SG5 or mu-SPL1 and control monoclonal antibodies dilutedin blocking buffer (1 μg/ml) for 60-90 minutes at room temperature. Thesections were then washed three times with the blocking buffer. Thebound monoclonal antibodies were detected with a goat anti-mouse IgG+IgM(H+L) F(ab′)²-peroxidase conjugates and the peroxidase substratediaminobenzidine (1 mg/ml, Sigma cat. No. D 5637) in 0.1 M sodiumacetate buffer pH 5.05 and 0.003% hydrogen peroxide (Sigma cat. No.H1009). The stained slides were counter-stained with hematoxylin andexamined under Nikon microscope.

In some cases, paraffin embedded formaldehyde-fixed tissues may be usedfor immunohistochemistry after appropriate antigen retrieval methodswere employed. One such antigen retrieval method is described in Manghamand Isaacson, Histopathology 35:129-33 (1999). Other methods of antigenretrieval and/or detection may be used by one skilled in the art.Results from similar experiments performed using frozen tissues or,where appropriate, fixed tissue with antigen retrieval and polyMICAdetection were performed. The binding of anti-LUCA19, anti-LUCA40 andanti-SPL1 antibodies to a variety of normal and cancer tissues wasassessed. In all cases, antibody binding in control fixed tissues wascorrelated with that of frozen tissues. The results from frozen tissueswere only used if the two did not match in the controls.

For convenience, a summary of the combined results of severalexperiments using frozen surgical tissue from different sources is shownbelow in Tables 1-7. Tables 1-4 summarize the distribution of LUCA19,LUCA40, SPL1, and SG5 antigens on normal human tissues. Tables 5-7summarize the distribution of LUCA19, LUCA40 and SPL1 antigens onvarious human tumor tissues.

TABLE 1 Distribution of LUCA19 antigen in normal human tissues TissueType Results Skin Negative Lung Negative Kidney Negative PancreasNegative except for 1+ staining on a few ducts Liver Negative ColonNegative Duodenum 1+ staining in basal epithelium

TABLE 2 Distribution of LUCA40 antigen in normal human tissues TissueType Results Skin Negative except +/− on a few small subcutaneousvessels Lung Negative Kidney Negative except for +/− on glomeruliPancreas Negative Liver Negative except for +/− on parenchyma ColonNegative except +/− over mucosa and muscularis Duodenum NegativeProstate Negative Ovary Negative Breast Negative except +/− overglandular structures

TABLE 3 Distribution of SPL1 antigen in normal human tissues Tissue TypeResults Skin Negative Lung Negative Kidney Negative Pancreas NegativeLiver Negative Colon 1+ focal staining on mucosa (50%) Duodenum 1+mucosal staining Prostate Negative Ovary Negative Breast Negative

TABLE 4 Distribution of SG5 antigen in normal human tissues Tissue TypeResults Skin +/− staining on epidermis and sweat glands Lung NegativeKidney Negative Pancreas Negative Liver +/− staining (parenchymal hue)Colon Negative Stomach Negative Prostate Negative Ovary Negative BreastNegative

TABLE 5 Distribution of LUCA19 antigen in human tumor tissues TissueType Results Prostate Negative on 4/4 tumors screened Colon 1-3+staining on 5/5 tumors screened Kidney Negative on 5/5 tumors screenedLung 1-2+ staining on 4/7 tumors screened and negative on 3/7 tumorsscreened Ovary Negative on 4/4 tumors screened Pancreas 2-3+ on 5/5tumors screened Breast Variable; 1-2+ on 1/3 tumors screened

TABLE 6 Distribution of LUCA40 antigen in human tumor tissues TissueType Results Prostate Variable; Negative to 1+ staining; 8 tumorsscreened Colon 1-3+ staining on 8/8 tumors screened Kidney +/− stromalstaining on 5/5 tumors screened Lung Variable; Negative to 1+ staining;8 tumors screened Ovary +/− stromal staining on 5/5 tumors screenedPancreas 1-2+ staining on 7/7 tumors screened Breast Negative on 3/4tumors screened; +/− on 1/4 tumors screened

TABLE 7 Distribution of SPL1 antigen in human tumor tissues Tissue TypeResults Prostate Negative on 6/6 tumors screened Colon 1-3+ apicalstaining on 7/9 tumors; Negative on 2/9 tumors screened Kidney Negativeon 5/5 tumors screened Lung Variable +/− to 1+ staining on 3/8 tumorsscreened; Negative on 5/8 tumors screened Ovary Negative on 5/5 tumorsscreened Pancreas 1-2+ on 6/6 tumors screened Breast Negative on 4/4tumors screened

Example 6 Immunocytochemistry Results

Monoclonal antibodies mu-LUCA19, mu-LUCA40 and mu-SPL1 were used to testreactivity with various cell lines from different types of tissues. Theresults were scored as ‘+’ for weak positive staining, ‘++’ for moderatepositive staining, ‘+++’ for strong positive staining and ‘−’ fornegative staining.

Immunohistochemistry results were obtained using CellArray™ technology,as described in WO 01/43869. Cells from different established cell lineswere removed from the growth surface without using proteases, packed andembedded in OCT compound. The cells were frozen and sectioned, thenstained using a standard IHC protocol.

Results of the binding of the mu-LUCA19, mu-LUCA40 and mu-SPL1antibodies to various established human normal and tumor cell lines arecompiled for convenience in Tables 8-10. The experiments represented inTable 8 include Live-cell ELISA, Sarcoma Array and CellArray™ bindingexperiments with mu-LUCA19 using the methods described herein. Theexperiments represented in Table 9 include Live-cell ELISA, SarcomaArray and Cell Array™ binding experiments with mu-LUCA40 using themethods described herein. The experiments represented in Table 10include Live-cell ELISA, Sarcoma Array and Cell Array™ bindingexperiments with mu-SPL1 using the methods described herein.

TABLE 8 Mu-LUCA19 binding Reactivity Reactivity Reactivity Cell SarcomaLive Cell Cell line ATCC# Organ Cell Type Array Array ELISA MDA361 HB-27Breast Adenocarcinoma − MDA175 HB-29 Breast Ductal carcinoma − MCF7HTB-22 Breast Adenocarcinoma − SKBR3 HTB-30 Breast Metastatic; pleural −− effusion adenocarcinoma BT474 HTB-20 Breast Ductal carcinoma − HUVECPrimary Endothelial Normal human ++ Cell adult HMEC CC-2251* BreastNormal mammary − epithelial SKOV3 HTB-77 Ovary Adenocarcinoma ++ +++ES-2 CRL-1978 Ovary Carcinoma + SKMES1 HTB-58 Lung Squamous − +carcinoma CA130 RAVEN Lung Small cell carcinoma +/− A549 CCL-185 LungCarcinoma + 9979 RAVEN Lung Lung cancer cell line − SW948 CCL-237 ColonColorectal − adenocarcinoma SW480 CCL-228 Colon Colorectal − +++adenocarcinoma HT-29 HTB-38 Colon Colorectal − + adenocarcinoma Colo205CCL-222 Colon Ascites colorectal +/− adenocarcinoma Hs700T HTB-147Pancreas Adenocarcinoma − HPAFII CRL-1997 Pancreas Adenocarcinoma ++ −AsPC-1 CRL-1682 Pancreas Adenocarcinoma + 9926 RAVEN PancreasAdenocarcinoma ++ SVT2 CCL-163.1 Embryo Fibroblast; SV40 − (Mouse)transformed Cos7 CRL-1651 Kidney SV40 transformed +/− (African GreenMonkey) RL65 CRL-10354 Lung (Rat) Epithelial cell − Calu3 HTB-55 LungAdenocarcinoma +++ Caki2 HTB-47 Kidney Clear cell carcinoma + A498HTB-44 Kidney Carcinoma +/− 786-O CRL-1932 Kidney Renal cell carcinoma+/− 293 CRL-1573 Kidney Transformed with − adenovirus5 DNA TDH RAVENProstate Prostate cancer cell − line PC3 CRL-1435 ProstateAdenocarcinoma − LNCaP CRL-1740 Prostate Carcinoma − DU145 HTB-81Prostate Adenocarcinoma +++ 22RV1 CRL-2505 Prostate Carcinoma − Hs746THTB-135 Stomach Carcinoma ++ N87 CRL-5822 Stomach Metastatic; liver ++gastric carcinoma SW872 HTB-92 Connective Liposarcoma ++ Tissue SW684HTB-91 Connective Fibrosarcoma ++ Tissue SK-UT-1 HTB-114 UterusLeiomyosarcoma ++ SK-LMS-1 HTB-88 Vulva Leiomyosarcoma ++ SK-ES-1 HTB-86Bone Ewing's sarcoma − RD-ES HTB-166 Bone Ewing's sarcoma +/− RD CCL-136Muscle Rhabdomyosarcoma − MG-63 CRL-1427 Bone Osteosarcoma − HT-1080CCL-121 Connective Fibrosarcoma ++ Tissue G-292 CRL-1423 BoneOsteosarcoma − A-204 HTB-82 Muscle Rhabdomyosarcoma + *CC-2251BioWhittaker

TABLE 9 Mu-LUCA40 binding Reactivity Reactivity Reactivity Cell SarcomaLive Cell Cell line ATCC# Organ Cell Type Array Array ELISA HMECCC-2251* Breast Normal mammary − epithelial HUVEC Primary EndothelialNormal human ++ Cell adult BT474 HTB-20 Breast Ductal carcinoma − MCF7HTB-22 Breast Adenocarcinoma − MDA175 HB-29 Breast Ductal carcinoma −MDA361 HB-27 Breast Adenocarcinoma − SKBR3 HTB-30 Breast Metastatic;pleural − − effusion adenocarcinoma 9979 RAVEN Lung Lung cancer cellline + A549 CCL-185 Lung Carcinoma + CA130 RAVEN Lung Small cellcarcinoma + Calu3 HTB-55 Lung Adenocarcinoma + SKMES1 HTB-58 LungSquamous − + carcinoma ES-2 CRL-1978 Ovary Carcinoma +++ SKOV3 HTB-77Ovary Adenocarcinoma + +++ 9926 RAVEN Pancreas Adenocarcinoma ++ AsPC-1CRL-1682 Pancreas Adenocarcinoma + HPAFII CRL-1997 PancreasAdenocarcinoma ++ − Hs700T HTB-147 Pancreas Adenocarcinoma + Colo205CCL-222 Colon Ascites colorectal + adenocarcinoma HT-29 HTB-38 ColonColorectal − + adenocarcinoma SW480 CCL-228 Colon Colorectal ++ +++adenocarcinoma SW948 CCL-237 Colon Colorectal − adenocarcinoma 293CRL-1573 Kidney Transformed with +/− adenovirus5 DNA 786-O CRL-1932Kidney Renal cell carcinoma + A498 HTB-44 Kidney Carcinoma + Caki2HTB-47 Kidney Clear cell carcinoma + Cos7 CRL-1651 Kidney SV40transformed ++ (African Green Monkey) RL65 CRL-10354 Lung (Rat)Epithelial cell − SVT2 CCL-163.1 Embryo Fibroblast; SV40 − (Mouse)transformed 22RV1 CRL-2505 Prostate Carcinoma − DU145 HTB-81 ProstateAdenocarcinoma − LNCaP CRL-1740 Prostate Carcinoma − PC3 CRL-1435Prostate Adenocarcinoma − TDH RAVEN Prostate Prostate cancer cell ++line Hs746T HTB-135 Stomach Carcinoma + N87 CRL-5822 Stomach Metastatic;liver ++ gastric carcinoma SW872 HTB-92 Connective Liposarcoma ++ TissueSW684 HTB-91 Connective Fibrosarcoma ++ Tissue SK-UT-1 HTB-114 UterusLeiomyosarcoma + SK-LMS-1 HTB-88 Vulva Leiomyosarcoma ++ SK-ES-1 HTB-86Bone Ewing's sarcoma − RD-ES HTB-166 Bone Ewing's sarcoma +/− RD CCL-136Muscle Rhabdomyosarcoma − MG-63 CRL-1427 Bone Osteosarcoma − HT-1080CCL-121 Connective Fibrosarcoma ++ Tissue G-292 CRL-1423 BoneOsteosarcoma − A204 HTB-82 Muscle Rhabdomyosarcoma + *CC-2251BioWhitaker

TABLE 10 Mu-SPL1 binding Reactivity Reactivity Reactivity Cell SarcomaLive Cell Cell line ATCC# Organ Cell Type Array Array ELISA HMECCC-2251* Breast Normal mammary − epithelial HUVEC Primary EndothelialNormal human ++ Cell adult BT474 HTB-20 Breast Ductal carcinoma − MCF7HTB-22 Breast Adenocarcinoma − MDA175 HB-29 Breast Ductal carcinoma −MDA361 HB-27 Breast Adenocarcinoma − SKBR3 HTB-30 Breast Metastatic;pleural − − effusion adenocarcinoma 9979 RAVEN Lung Lung cancer cellline + A549 CCL-185 Lung Carcinoma + CA130 RAVEN Lung Small cellcarcinoma + Calu3 HTB-55 Lung Adenocarcinoma + SKMES1 HTB-58 LungSquamous − + carcinoma ES-2 CRL-1978 Ovary Carcinoma +++ SKOV3 HTB-77Ovary Adenocarcinoma ++ +++ 9926 RAVEN Pancreas Adenocarcinoma ++ AsPC-1CRL-1682 Pancreas Adenocarcinoma ++ HPAFII CRL-1997 PancreasAdenocarcinoma ++ (75%) − Hs700T HTB-147 Pancreas Adenocarcinoma +/−Colo205 CCL-222 Colon Ascites colorectal +/− adenocarcinoma HT-29 HTB-38Colon Colorectal +/− + adenocarcinoma SW480 CCL-228 Colon Colorectal +++++ adenocarcinoma SW948 CCL-237 Colon Colorectal + adenocarcinoma 293CRL-1573 Kidney Transformed with +/− adenovirus5 DNA 786-O CRL-1932Kidney Renal cell carcinoma + A498 HTB-44 Kidney Carcinoma + Caki2HTB-47 Kidney Clear cell carcinoma ++ Cos7 CRL-1651 Kidney SV40transformed ++ (African Green Monkey) RL65 CRL-10354 Lung (Rat)Epithelial cell − SVT2 CCL-163.1 Embryo Fibroblast; SV40 − (Mouse)transformed 22RV1 CRL-2505 Prostate Carcinoma − DU145 HTB-81 ProstateAdenocarcinoma +/− LNCaP CRL-1740 Prostate Carcinoma − PC3 CRL-1435Prostate Adenocarcinoma − TDH RAVEN Prostate Prostate cancer cell ++line Hs746T HTB-135 Stomach Carcinoma + N87 CRL-5822 Stomach Metastatic;liver ++ gastric carcinoma SW872 HTB-92 Connective Liposarcoma + TissueSW684 HTB-91 Connective Fibrosarcoma ++ Tissue SK-UT-1 HTB-114 UterusLeiomyosarcoma + SK-LMS-1 HTB-88 Vulva Leiomyosarcoma ++ SK-ES-1 HTB-86Bone Ewing's sarcoma − RD-ES HTB-166 Bone Ewing's sarcoma − RD CCL-136Muscle Rhabdomyosarcoma − MG-63 CRL-1427 Bone Osteosarcoma − HT-1080CCL-121 Connective Fibrosarcoma ++ Tissue G-292 CRL-1423 BoneOsteosarcoma − A204 HTB-82 Muscle Rhabdomyosarcoma + *CC-2251BioWhitaker

Results of the binding of the mu-LUCA19, mu-LUCA40 and mu-SPL1antibodies to various established tumor cell lines from the NationalCancer Institute (NCI) are compiled for convenience in Table 11 usingthe methods described herein.

TABLE 11 NCI Cell Line Array LUCA19 LUCA40 SPL1 Cell Line Tumor OriginHistologic Type Reactivity Reactivity Reactivity CRF-CEM PeripheralAcute lymphoblastic − − − blood leukemia HL-60(TB) Procyelocyticleukemia − − − K-562 Pleural effusion Chronic myelogenous − − − leukemiaMOLT-4 Peripheral Acute lymphoblastic − − − blood leukemia RPMI-8226Peripheral Multiple myeloma − − − blood SR Immunoblastic large − − −cell lymphoma A549 Lung Carcinoma + + + EKVX Lung Adenocarcinoma − − −HOP-62 Lung Adenocarcinoma +++ +++ +++ HOP-92 Lung Large cell, + + ++undifferentiated NCI-H226 Lung Squamous cell + ++ ++ NCI-H23 LungAdenocarcinoma +/− − + NCI-H322M Lung Bronchi alveolar − − − carcinomaNCI-H460 Lung Non-small cell − − − NCI-H522 Lung Adenocarcinoma ++ ++ ++Colo205 Colon Ascites colorectal +/− +/− + (50%) adenocarcinoma HCC-2998Colon Carcinoma ++ ++ +++ HCT-116 Colon Carcinoma ++ ++ ++ HCT-15 ColonAdenocarcinoma + +/− + HT29 Colon Colorectal − − + adenocarcinoma KM12Colon Adenocarcinoma + ++ ++ SW-620 Colorectal Metastatic; lymph ++ +++++ adenocarcinoma node SF-268 CNS Anaplastic ++ ++ ++ astrocytomaSF-295 CNS Glioblastoma + + + multiform SF-539 CNS Gliosarcoma ++ ++ ++SNB-19 CNS Glioblastoma ++ ++ ++ SNB-75 CNS Astrocytoma +/− +/− +/− U251CNS Glioblastoma +++ ++ ++ LOX IMVI Lymph node Malignant amelanotic + +++ metastasis melanoma MALME-3M Lung metastasis Malignant melanoma − − +M14 Amelanotic melanoma − − − SK-MEL-2 Skin metastasis- Malignantmelanoma − − − thigh SK-MEL-28 Malignant melanoma − − − SK-MEL-5Axcillary node Malignant melanoma − − − metastasis UACC--257 Malignantmelanoma + + + UACC-62 Malignant melanoma − − − IGR-OV1 OvaryCystoadenocarcinoma − − − OVCAR-3 Ovary Adenocarcinoma ++ ++ (75%) ++OVCAR-4 Ovary Adenocarcinoma + + + OVCAR-5 Ovary Adenocarcinoma +++ +++++ OVCAR-8 Ovary Adenocarcinoma +++ + ++ SKOV3 Ovary Adenocarcinoma++ + + PC3 Prostate Adenocarcinoma − − − DU145 Prostate Adenocarcinoma −− − 786-O Kidney Renal cell carcinoma +/− + + A498 Kidney Carcinoma+/− + + ACHN Kidney Carcinoma +++ ++ ++ CAKI-1 Kidney Adenocarcinoma ++++ ++ SN12C Kidney Carcinoma ++ ++ +++ TK-10 Kidney Carcinoma + + +UO-31 Kidney Carcinoma ++ ++ ++ MCF-7 Breast Adenocarcinoma − − −NCI-ADR-RES Breast Metastatic; Pleural + + ++ effusion adenocarcinomaMDA-MB-231 Breast Metastatic; Pleural ++ ++ +++ effusion adenocarcinomaHs 578T Breast Ductal carcinoma + +/− + MDA-MB-435 Breast Metastatic;Pleural − − − effusion adenocarcinoma BT-549 Breast Metastatic; Lymph+++ +++ +++ node; Inflitrating ductal carcinoma T-47D Breast Metastatic;Pleural − − − effusion; Ductal carcinoma

Monoclonal antibodies mu-LUCA19, mu-LUCA40 and mu-SPL1 were used to testreactivity with glioma-derived cell lines. Immunocytochemistry resultswere obtained using similar protocols as described above for theCellArray™ technology. The glioma-derived cell lines were removed fromthe growth surface without using proteases, packed and embedded in OCTcompound. The cells were frozen and sectioned, then stained using astandard IHC protocol. Mu-LUCA19 was positive on 17 out of 25glioma-derived cell lines screened. Staining intensity ranged from ± to3+ staining. Mu-LUCA40 was positive on 18 out of 25 glioma-derived celllines screened. Staining intensity ranged from ± to 3+ staining.Mu-SPL-1 was positive on 14 out of 25 glioma-derived cell linesscreened. Staining intensity ranged from ± to 3+ staining.

Example 7 Isolation and Characterization of Antigen Ag-SPL1, Ag-LUCA19,Ag-SG5 and Ag-LUC40

To identify the antigen to which SPL1, LUCA19, SG5 and LUCA40 werereactive, an immunoprecipitation (Ippt) experiment was performed. Forimmunoprecipitation, thirty 175 cm² flasks of SW480 cells were lysedwith 30 ml of lysis buffer. The lysis buffer consisted of Hanks balancedsalt solution (HBSS+) fortified with 2% Triton X-100, protease inhibitorcocktail (1 tablet per 5 ml lysis buffer of complete mini EDTA freeprotease cocktail from Roche Molecular Biochemicals), 0.1% sodium azide,and 2 mM PMSF. The cell lysate was clarified at 24,000×g for 30 minutesat 4° C. before being passed over a column consisting of 1 ml Protein G(Amersham Pharmacia). The pre-cleared SW480 lysate was then incubatedwith Protein G absorbed mu-SPL1, mu-LUCA19, mu-SG5 or mu-LUCA40 (10₄gmu-SPL1, mu-LUCA19, mu-SG5or mu-LUCA40 was pre-incubated for 30 minutesat room temp with 5 μl Protein G) for 2 hours at 4° C. The beads (boththe pre-clear Protein G beads and the Protein G absorbed mu-SPL1,mu-LUCA19, mu-SG5 or mu-LUCA40 beads) were then washed three times withlysis buffer before elution with 30 μl SDS sample buffer (3% SDS, 20%Glycerol, 10 mM DTT, 2% Bromophenol blue, 0.1M Tris, pH8.0). 25 μl ofthe eluate was then resolved by SDS-PAGE and visualized throughCoomassie staining. 5 μl of the eluate was resolved by SDS-PAGE andfurther transferred to nitrocellulose for western blotting.

The blot was then probed with mu-SPL1, mu-LUCA19, mu-SG5 or mu-LUCA40and developed using a Western Blotting Kit (Invitrogen Cat. No. WB7103)to confirm antigen recognition. By western blotting the mu-SPL1,mu-LUCA19, mu-SG5 or mu-LUCA40 and mouse IgG eluate against mu-SPL1,mu-LUCA19 mu-SG5 or mu-LUCA40, a protein unique to the mu-SPL 1,mu-LUCA19, mu-SG5 or mu-LUCA40 eluate was observed. Stained proteinbands from the NuPAGE gel were excised using clean scalpel blades andare placed in clean Eppendorf tubes. Excised bands were stored at −20°C. until used for protein identification by mass spectrometry.

Example 8 Characterization of the Antigen to which mu-SPL1, mu-LUCA19,mu-SG5 or mu-LUCA40 Binds Using Tandem Mass Spectrometry (MS/MS)

The antigens to which mu-SPL1, mu-LUCA19, mu-SG5 and mu-LUCA40 bind wereisolated as described in Example 7 and subjected to Tandem massspectroscopy according to the method of Kane et al. J Bio Chem. 2002Jun. 21; 277(25):22115-8, Epub May 06. Proteins are separated bySDS-PAGE, and the gel is stained with the colloidal Coomassie Bluereagent (Invitrogen). Proteins of interest are digested in gel withtrypsin. The tryptic peptides are sequenced by microcapillary liquidchromatography MS/MS on an ion trap mass spectrometer (Thermo-FinniganLCDQ DECA XP), as described in Wu et al., Nature 405:477-482 (2000).

Alternatively, other commonly known methods of mass spectrometry, suchas MALDI mass spectrometry, are also used in the practice of thisinvention. The results from the mass spectroscopy analysis showed thatthe antigen to which mu-SPL1, mu-LUCA19, mu-SG5 and mu-LUCA40 was EphA2.

Example 9 Sandwich ELISA for Epitope Mapping of mu-SPL1, mu-LUCA19,mu-SG5 and mu-LUCA40

The specific epitope on EphA2 to which mu-SPL1, mu-LUCA19, mu-SG5 andmu-LUCA40 bind was assessed using a competitive sandwich ELISA. 50μl/well of 10 μg/ml concentration of mu-SPL1, mu-LUCA19, mu-SG5 ormu-LUCA40 were diluted in HBSS (Hank's Balanced Salt Solution with outsodium bicarbonate and phenol red) and allowed to bind to 96-wellMaxisorb micro-titer plates for 2 hours at room temperature. Coatedplates are then blocked for 30 minutes with HBSS containing 1% BSA(W/V). HT-29 cells were lysed as described in example 7. The lysate isthen applied to the blocked micro-titer wells at a volume of 150 μl perwell. The lysates are allowed to incubate for 1 hour at roomtemperature. After the incubation, the plate is then washed thoroughlywith blocking buffer. Biotinylated mu-SPL1, mu-LUCA19, mu-SG5 andmu-LUCA40, at a concentration of 50 μl/well, 1 μg/ml, are added into themicro-titer wells and allowed to incubate for 1 hour at room temp.Biotinylated mu-SPL1, mu-LUCA19, mu-SG5 and mu-LUCA40 were prepared byincubating 1 μl of 200 mg/ml Sulfo-NHS-LC-Biotin diluted in DMSO forevery 1 mg of purified antibody for 2 hours at room temperature. Thebiotinylated antibodies are then quenched with a final concentration of0.1M Tris, pH 7.4 for 30 minutes at room temperature. The biotinylatedantibody is then separated from free biotin by concentrating through a30 kDa molecular weight cut-off filter. After the 1 hour incubation ofthe biotinylated antibodies, the plates are washed thoroughly with HBSS.Horseradish peroxidase conjugated streptavidin (SA-HRP) is diluted intoHBSS at a concentration of 1:10,000. The diluted SA-HRP is added intothe washed micro-titer wells at 50 μl/well and allowed to incubate for30 minutes at room temp. After the incubation of SA-HRP, the plate isfinally washed thoroughly with HBSS and developed with the addition of100 μl/well of 1-Step TMB substrate for a color change reaction. Thedevelopment is stopped with the addition of 100 μl/well of 1M phosphoricacid diluted in Milli-Q water. The developed plate is then analyzed witha ThermoMax micro-titer plate reader at 450 nm.

From the results of the competitive sandwich ELISA, we can conclude thatmu-SPL1, mu-LUCA19, mu-SG5 and mu-LUCA40 represent three antibodiesbinding EphA2 through two distinctive epitopes, one represented bymu-LUCA40, and the second represented by mu-SPL1. mu-LUCA19 seems toshare, but not directly overlap the epitope represented by mu-SPL1. Theoverlap does not extend into the epitope represented by the mu-LUCA40epitope.

Example 10 Further Characterization of mu-LUCA19 and mu-SG5

EphA2 stimulation in G55 cells, a glioma cell line that expressesendogenous EphA2, was studied using mu-LUCA19 and mu-SG5. The cells weretreated with either mu-LUCA19 or mu-SG5 in increasing concentrations upto 30 μg/ml and the phosphotyrosine content of immunoprecipitated EphA2was measured by Western blot analysis with phosphotyrosine-specificantibodies (4G10, Upstate Cell Signaling Solutions, New York). Nodifference in phosphotyrosine content in EphA2 immunoprecipitated fromG55 cells treated with either mu-LUCA19 or mu-SG5 was seen. ControlWestern blots for total EphA2 content was performed to ensure that thetotal EphA2 protein that was immunoprecipitated was the same for eachtreatment condition. Similar results were seen using A549 cells andmu-LUCA19 and mu-SG5 antibodies. These results indicate that treatmentof cells expressing endogenous EphA2 with either mu-LUCA19 or mu-SG5does not increase or decrease the phosphotyrosine content of EphA2.

Example 11 Effect of mu-SPL1, mu-LUCA19 and mu-LUCA40 on Cancer CellLines

The ability of the antibodies to reduce cell number in vitro when grownas a monolayer can be assessed using cell monolayers grown in thepresence or absence of varying amounts of test or control purifiedantibody and the change in cell number assessed using MTT. MTT is a dyethat measures the activity of mitochondrial enzymes and correlates withrelative viable cell number. Cells of interest were plated and grown inF12/DMEM (1:1) growth medium supplemented with 10% fetal bovine serum in96 well plates. The cell lines were plated in triplicate wells of a96-well dish at a density range of 1500-2500 cells/well. Immediatelyafter plating, mu-SPL1, mu-LUCA19 or mu-LUCA40 was added. The cells wereincubated at 37° C. in a humidified incubator at 5% CO2/air for 5 days.At the end of the assay, MTT was dissolved in PBS (5 mg/ml) and addeddirectly to wells at 1:10 dilution. Plates were placed back in incubatorfor 4 hours. After the incubation, medium was removed and 100 μl DMSOwas added to solubilize the MTT precipitate. Plates were read at O.D.540 nm. In these experiments, mu-LUCA40 inhibited growth of 786-O, A549,Caki-2, ES-2, SKMES-1, and SKOV3 cell lines, and SPL1 and LUCA19 didnot.

Example 12 Internalization of mu-LUCA19, mu-SG5 and mu-LUCA40 andToxin-Conjugated Anti-Mouse IgG

Mab-ZAP (Advanced Targeting Systems, San Diego, Calif.) is an anti-mouseIgG conjugated to saporin, a toxin that inhibits protein synthesis. Thistoxin is impermeable to the cell membrane. If a monoclonal antibody isbound to a cell-surface antigen that is internalizable, thetoxin-conjugate can bind to the bound monoclonal and, thereby, beinternalized and eventually kill the cell. Being dependent uponinternalization for demonstration of toxic activity, the Mab-ZAP canserve to evaluate whether or not a given surface antigen will serve as asuitable target for any toxin that is dependent upon internalization toexpress cell toxic effects. As such, the Mab-ZAP serves as a model forsuch internalization-dependent toxins such as maytansinoids andcalicheamicin.

For testing the internalization of mu-LUCA19 and mu-LUCA40 and saporinconjugated anti-mouse IgG by tumor cells and effect of killing the tumorcells after internalization of saporin, human glioma cells, G130 wereremoved from stock flasks with 10 mM EDTA and centrifuged. Cells wereresuspended at 50,000/ml in appropriate medium and 100 μl plated perwell in 96 well plates. Antibody mu-SPL1, mu-LUCA19 or mu-LUCA40 wasadded immediately to appropriate wells as a 10× concentrate, to make afinal concentration of 10 ug/ml. After 15 minutes at room temperatureMab-ZAP (Cat. #IT-04, Advanced Targeting Systems, San Diego Calif.) wasadded to appropriate wells as 10× concentrate, to make finalconcentrations from 0.001 nM to 10 nM. After 4 days growth, MTT wasadded (stock 5 mg/ml PBS, 1:10 dilution in well) for 4 hrs at 37° C. Themedium was then removed from all wells and 100 μl/well DMSO was added.The plates were gently swirled to solubilize the blue MTT precipitateand the plates were read at O.D. 540 nm.

There was a decrease in MTT staining in G130 cells in the presence ofmu-LUCA19 and mu-LUCA40 as compared to staining in the absence of thoseantibodies. This indicates that the growth of G130 cells was inhibitedin the presence of mu-LUCA19 and mu-LUCA40 and Mab-ZAP and these resultsare indicative of mu-LUCA19 and mu-LUCA40 and toxin-conjugatedanti-mouse IgG were internalized in G130 cells. Similar results wereseen using ES-2 cells, an ovarian carcinoma cell line, and mu-SG5antibodies. There was a decrease in MTT staining in ES-2 cells in thepresence of mu-SG5 as compared to staining in the absence of thisantibody. This indicates that the growth of ES-2 cells was inhibited inthe presence of mu-SG5 and Mab-ZAP. This result is indicative of mu-SG5and toxin-conjugated anti-mouse IgG were internalized in ES-2cells.

Example 13 Efficacy of Anti-EphA2 Antibody SPL1 and LUCA40 with HumanCells in Nude Mice

Human tumor cells were grafted under the kidney capsule in nude (nu/nu)mice. Three tumor cell lines were used. The ES-2 and SKOV3-3 ovariantumor-derived cell lines, and the Caki-2 kidney tumor-derived cell lineswere obtained from the ATCC. In some studies both kidneys receivedxenografts. For the treated animals, grafts were made in the kidneycapsule (500k cells in collagen gel). Anti-EphA2 monoclonal antibodySPL1 or LUCA40 was injected intraperitoneally at a concentration of 50mg/kg and a volume of 0.01 mL/g body weight. Dosing was initiated on Day2 following implantation, and doses of SPL1, LUCA40 or PBS wereadministered three times weekly as single rapid injections. Control micewere injected with PBS only. Three days after the final injection, theanimals were euthanized and the kidneys with grafts were examined.

The amount of human DNA in the tumors was quantitated using real-timePCR on an Applied Biosystems (Foster City, Calif.) SDS7000 system, withprimers and probe specific for the human ribosomal gene RPL19 accordingto published methods. Each tumor sample was analyzed in triplicate PCRreactions and average DNA concentrations were determined. Average DNAconcentration and standard error of the mean was determined for eachgroup of tumor samples. Statistical significance was determined usingthe Student's T-test (two-tailed, type 1). Tumor growth inhibition wascalculated as the absolute value of [(average tumor volume of treatedgroup/average tumor volume of PBS control group)×100]−100.

In data not shown, SPL1 appeared on visual and manual examination toreduce the size of tumors, relative to controls, using cell lines Caki-2and ES-2. Similar results were seen in LUCA40 treated mice implantedwith Caki-2 tumors. DNA quantitation of the Caki-2 tumors from micetreated with LUCA40 antibodies confirmed that the LUCA40 treated micehad smaller tumors relative to controls. Table 12 shows other sub-renalcapsule xenograft study designs with SPL1 antibodies and DNAquantitation results from these studies.

TABLE 12 SPL1 Sub-Renal Capsule Xenograft Model Study Design and ResultsCell Line MAb dose No. animals/ Studied (mg/kg) group % TGI* ES-2 50 578.3 p = 0.006 ES-2 50 6 61 p = 0.026 SKOV3-3 50 6 70 p = 0.005 *TGI =tumor growth inhibition vs. PBS control group; p-values were determinedby the Student's T-test, unpaired.

Example 14 Antitumor Efficacy of SPL1 in a Subcutaneous Model of HumanOvarian Tumors

This study was designed to test the dose-responsive anti-tumor data forSPL1 antibody in a subcutaneous model of ovarian cancer and lung cancer.

The ES-2 ovarian tumor-derived cell line was re-derived from arapid-growing tumor identified in a pilot subcutaneous tumor modelstudy. A549 lung tumor-derived cells were obtained from the ATCC.Cultured cells were trypsinized, washed in media, spun down andresuspended in media at 100 million cells per milliliter of media (5million cells per 0.05 mL volume), then mixed in an equal volume ofMatrigel® for a final injection volume of 0.1 mL. Female CRL. nu/nuhomozygous mice were used. Cells were inoculated by subcutaneousinjection in the back of the neck of the mice. Dosing was initiatedeither on the day of implantation or when tumors were established andmeasurable. For Day 0 dosing, tumors were implanted in the morning andanimals dosed in the afternoon of the same day. For established tumors,animals were randomized among groups as follows: tumor volumes weredetermined, animals were sorted by tumor volume, the mean was determinedand the appropriate number of animals (12 to 15 per group depending onthe model) was selected above and below the mean, removing from thestudy those with small or large tumors. The remaining animals wererandomized by ear tag number into treatment and control groups. Therandom distribution of the final groups was confirmed by T-test (p>0.1was considered randomized).

For each treatment dose group, SPL1 was diluted in PBS at an appropriateconcentration dose (50 mg/kg for ES2 cells study and a concentrationrange of 10mg/kg, 30 mg/kg and 100 mg/kg for A549 cells study) and PBScontrol and administered at a volume of 0.01 mL/g body weight. Doses ofSPL1 or PBS were administered twice weekly as single rapid injectionsinto the intraperitoneal cavity. Control mice were injected with PBSonly. Dosing was initiated in groups of 12-15 mice when tumors reachedthe appropriate size, depending on the study.

Tumors were allowed to grow for approximately 6 days prior to initialtumor measurement. Tumors were subsequently measured twice weekly bydigital caliper in three dimensions, and tumor volume was calculated asone-half the product of the three measurements. Tumor volume over timewas the primary endpoint for all studies. Clinical observations weremade daily. Body weight was determined for each animal twice weekly.

Average tumor volumes and standard error of the mean were determined foreach group at each measurement. Statistical significance was determinedusing the Student's T-test (two-tailed, type 1). Tumor growth inhibitionwas calculated as the absolute value of [(average tumor volume oftreated group/average tumor volume of PBS control group)×100]−100. Table13 shows subcutaneous xenograft study designs and results.

TABLE 13 SPL1 SC Xenograft Model Study Design and Results Cell Line MAbdose Dose No. animals/ Studied (mg/kg) Regimen group % TGI* ES-2 50Start dosing 12 44.9% Day 0 p = 0.045 Day 18 PBS Start dosing 12 NA Day0 50 Start dosing 12-15 47.6% when tumors p = 0.012 reach 100-150 mm³Day 21 PBS Start dosing 12-15 NA when tumors reach 100-200 m³ A549 10Start dosing 15 67.5% when tumors p = <0.001 reach 100 mm³ Day 34 30Start dosing 15 82.2% when tumors p = <0.001 reach 100 mm³ Day 34 100 Start dosing 15 80.4% when tumors p = <0.001 reach 100 mm³ Day 34 PBSStart dosing 15 NA when tumors reach 100 mm³ *TGI = tumor growthinhibition vs. PBS control group.

As shown in FIG. 1, a subset of SPL1 treated A549 tumor xenografts wasfound to re-grow slowly after cessation of dosing, with tumor re-growthdefined as an increase in volume of two-fold or more after cessation ofdosing. 7 out of 16 mice in the 100 mg/kg treatment group had tumorregrowth, while 9 out of 16 mice in the 100 mg/kg treatment groupremained tumor free after cessation of dosing. Similarly, 3 out of 15mice in the 30 mg/kg treatment group had tumor regrowth, while 12/15mice in the 30 mg/kg remained tumor free after cessation of dosing. Thegraphed results in FIG. 1 after the “stop dosing” point represent onlythe tumor size from mice that had tumor regrowth.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application. Allpublications, patents and patent applications cited herein are herebyincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent or patent applicationwere specifically and individually indicated to be so incorporated byreference.

1. An isolated nucleic acid sequence that encodes an EphA2 antibody,wherein said antibody specifically binds to EphA2 and comprises thethree complementarity determining regions from the heavy chain and thethree complementarity determining regions from the light chain ofantibody LUCA19 produced by the cell line having ATCC No. PTA-5070. 2.The nucleic acid of claim 1, wherein the nucleic acid is operably linkedto a promoter.
 3. The nucleic acid of claim 2, wherein the promoter andthe nucleic acid are contained in an expression vector.
 4. The nucleicacid of claim 1, wherein said EphA2 antibody is an antigen-bindingfragment selected from the group consisting of a Fab, a Fab', a F(ab')₂and a Fv; wherein said antigen binding fragment retains the bindingspecificity of antibody LUCA19 produced by the cell line having ATCC No.5070.
 5. A cell line transfected, transformed, or infected with a vectorcontaining a nucleic acid of claim
 1. 6. A method for producing asubstantially purified immunoglobulin, or an antigen binding fragmentthereof, comprising the steps of: a. Growing a cell line transformedwith the nucleic acid of claim 1 under conditions in which theimmunoglobulin or antigen binding fragment is expressed; and b.Harvesting the expressed immunoglobulin or fragment.
 7. The method ofclaim 6, wherein the cell line is a hybridoma.
 8. The nucleic acid ofclaim 1, wherein said EphA2 antibody is a humanized antibody.
 9. Thenucleic acid of claim 8, wherein said EphA2 antibody is anantigen-binding fragment selected from the group consisting of a Fab, aFab', a F(ab')₂ and a Fv; wherein said antigen binding fragment retainsthe binding specificity of antibody LUCA19 produced by the cell linehaving ATCC No.
 5070. 10. An isolated nucleic acid sequence that encodesan EphA2 antibody, wherein said antibody specifically binds to EphA2 andcomprises the three complementarity determining regions from the heavychain and the three complementarity determining regions from the lightchain of antibody SPL1 produced by the cell line having ATCC No.PTA-6059.
 11. The nucleic acid of claim 10, wherein the nucleic acid isoperably linked to a promoter.
 12. The nucleic acid of claim 11, whereinthe promoter and the nucleic acid are contained in an expression vector.13. The nucleic acid of claim 10, wherein said EphA2 antibody is anantigen-binding fragment selected from the group consisting of a Fab, aFab', a F(ab')₂ and a Fv; wherein said antigen binding fragment retainsthe binding specificity of antibody SPL1 produced by the cell linehaving ATCC No.
 6059. 14. A cell line transfected, transformed, orinfected with a vector containing a nucleic acid of claim
 10. 15. Amethod for producing a substantially purified immunoglobulin, or anantigen binding fragment thereof, comprising the steps of: a. Growing acell line transformed with the nucleic acid of claim 10 under conditionsin which the immunoglobulin or antigen binding fragment is expressed;and b. Harvesting the expressed immunoglobulin or fragment.
 16. Themethod of claim 15, wherein the cell line is a hybridoma.
 17. Thenucleic acid of claim 10, wherein said EphA2 antibody is a humanizedantibody.
 18. The nucleic acid of claim 17, wherein said EphA2 antibodyis an antigen-binding fragment selected from the group consisting of aFab, a Fab', a F(ab')₂ and a Fv; wherein said antigen binding fragmentretains the binding specificity of antibody SPL1 produced by the cellline having ATCC No.
 6059. 19. An isolated nucleic acid sequence thatencodes an EphA2 antibody, wherein said antibody specifically binds toEphA2 and comprises the three complementarity determining regions fromthe heavy chain and the three complementarity determining regions fromthe light chain of antibody LUCA40 produced by the cell line having ATCCNo. PTA-6056.
 20. The nucleic acid of claim 19, wherein the nucleic acidis operably linked to a promoter.
 21. The nucleic acid of claim 20,wherein the promoter and the nucleic acid are contained in an expressionvector.
 22. The nucleic acid of claim 19, wherein said EphA2 antibody isan antigen-binding fragment selected from the group consisting of a Fab,a Fab', a F(ab')₂ and a Fv; wherein said antigen binding fragmentretains the binding specificity of antibody LUCA40 produced by the cellline having ATCC No.
 6056. 23. A cell line transfected, transformed, orinfected with a vector containing a nucleic acid of claim
 19. 24. Amethod for producing a substantially purified immunoglobulin, or anantigen binding fragment thereof, comprising the steps of: a. Growing acell line transformed with the nucleic acid of claim 10 under conditionsin which the immunoglobulin or antigen binding fragment is expressed;and b. Harvesting the expressed immunoglobulin or fragment.
 25. Themethod of claim 24, wherein the cell line is a hybridoma.
 26. Thenucleic acid of claim 19, wherein said EphA2 antibody is a humanizedantibody.
 27. The nucleic acid of claim 26, wherein said EphA2 antibodyis an antigen-binding fragment selected from the group consisting of aFab, a Fab', a F(ab')₂ and a Fv; wherein said antigen binding fragmentretains the binding specificity of antibody LUCA40 produced by the cellline having ATCC No.
 6056. 28. An isolated nucleic acid sequence thatencodes an EphA2 antibody, wherein said antibody specifically binds toEphA2 and comprises the three complementarity determining regions fromthe heavy chain and the three complementarity determining regions fromthe light chain of antibody SG5 produced by the cell line having ATCCNo. PTA-7356.
 29. The nucleic acid of claim 28, wherein the nucleic acidis operably linked to a promoter.
 30. The nucleic acid of claim 29,wherein the promoter and the nucleic acid are contained in an expressionvector.
 31. The nucleic acid of claim 28, wherein said EphA2 antibody isan antigen-binding fragment selected from the group consisting of a Fab,a Fab', a F(ab')₂ and a Fv; wherein said antigen binding fragmentretains the binding specificity of antibody LUCA19 produced by the cellline having ATCC No.
 5070. 32. A cell line transfected, transformed, orinfected with a vector containing a nucleic acid of claim
 28. 33. Amethod for producing a substantially purified immunoglobulin, or anantigen binding fragment thereof, comprising the steps of: a. Growing acell line transformed with the nucleic acid of claim 28 under conditionsin which the immunoglobulin or antigen binding fragment is expressed;and b. Harvesting the expressed immunoglobulin or fragment.
 34. Themethod of claim 33, wherein the cell line is a hybridoma.
 35. Thenucleic acid of claim 28, wherein said EphA2 antibody is a humanizedantibody.
 36. The nucleic acid of claim 35, wherein said EphA2 antibodyis an antigen-binding fragment selected from the group consisting of aFab, a Fab', a F(ab')₂ and a Fv; wherein said antigen binding fragmentretains the binding specificity of antibody SG5 produced by the cellline having ATCC No. 7356.